Dep2 and its uses in major depressive disorder and other related disorders

ABSTRACT

The present invention relates to DEP2, as well as other proteins, and their uses in connection with the treatment of major depression or related disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 11/509,296,filed on Aug. 24, 2006, which is a continuation-in-part of U.S. patentapplication Ser. No. 11/412,184, filed on Apr. 26, 2006, the entirecontents of all of which are fully incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 7, 2014, isnamed 2014_(—)10_(—)08_(—8108)USD1-SEQ-LIST.txt and is 322,598 bytes insize.

BACKGROUND INFORMATION

Mood disorders, of which major depressive disorder is the most common,affect one person in five during their lifetime. The World HealthOrganization estimates that depression is currently the fourth mostimportant worldwide cause of disability-adjusted life year loss, andthat it will become the second most important cause by 2020 (See, MurrayC J L and Lopez A D, The Global Burden of Disease: A ComprehensiveAssessment of Mortality and Disability From Disease, Injuries, and RiskFactors in 1990 and Projected to 2020, volume 1. World HealthOrganization. Cambridge Mass.: Harvard University Press. 1996.).Pharmaceutical treatment of depression is frequently inadequate. Inrandomized clinical trials of the current best treatments, one-third ofpatients or more do not achieve remission, even after several months oftreatment (See, Journal of the American Medical Association, 286:2947-55(2001); Biological Psychiatry, 48:894-901 (2000)). Even when today'sdrugs do help patients achieve remission from their depression, theonset of action is over a period of weeks and there appears to be anincreased risk of suicide during initial antidepressant therapy,although this risk may be less than that just prior to therapyinitiation (See, Neuropsychopharmacology, 31:473-492 (2006)). Further,there are high recurrence rates—approximately 85% of patients whoachieve remission will suffer another episode of major depression (See,American Journal of Psychiatry, 156:1000-6 (1999)). Finally, currentlyavailable antidepressants are associated with side effects that leadsome patients to stop taking their medications at risk of sinking back(further) into depression, and to morbidity in others (See, New EnglandJournal of Medicine, 353: 1819-34 (2005)).

The currently available antidepressants work primarily by increasing theactivity of certain neurotransmitters, serotonin and norepinephrine, insynapses. Some medications (such as monoamine oxidase inhibitors)inhibit the degradation of these molecules, others (such as selectiveserotonin reuptake inhibitors and dual serotonin/norepinephrine reuptakeinhibitors) decrease removal of neurotransmitters from the synapticspace, and some medications (such as receptor antagonists) stimulatenorepinephrine release or inhibit negative feedback of serotoninsignaling. Because these medications are all based on a singleprinciple, the strength and range of their efficacy is similar. Theimprovements of the last half century have involved the development ofsafer and more tolerable drugs. However, despite this, today's drugs areneither completely safe nor completely tolerable for many patients.

Thus, there is considerable need for new drugs that are effective in abroader range of patients (particularly for patients whose depression isresistant to available pharmaceuticals), that have a faster onset ofaction, that are safer and more tolerable, or that complement theefficacy of existing drugs. It is possible, but unlikely, that furtherimprovement in any of these dimensions will be achieved throughdevelopment of additional serotonergic or noradrenergic agents.Therefore, alternative pharmacological approaches must be developed andpursued.

Part of the challenge in developing new drugs lies in the complexity ofdemonstrating efficacy of a major depression treatment. For example, thedevelopment of novel antidepressants is constrained by the limitedunderstanding of depression's etiology. Because of this, there arerelatively few pharmacological targets that can be considered forantidepressant development. Thereupon, there is a need for theidentification of drug targets for depression. Genetic linkage can opennew windows for the development of novel depression drug targets.Specifically, if a genetic variant is identified as being linked todepression in families, the gene in which that variant occurs is likelyto be involved in the etiology of disease. Such a gene can be a targetfor the development of novel antidepressants. Additionally, such a genecan lead to the identification of previously unknown physiologicalpathways that may be modulated for effective therapy of depression.

Several genes have been identified or proposed as factors for depressionor related phenotypes. Among these, most have been associated withdisease in population studies of candidate genes selected on the basisof existing hypotheses about the etiology of depression. Many of thesegenes relate to serotonin or norepinephrine. Examples include: (1)associations of a HTR1A (serotonin receptor 1A) promoter variant withdepression, suicide, bipolar disorder, panic disorder with agoraphobia,neuroticism and anti-depressant response; (2) associations of the HTT(serotonin transporter) promoter short allele with depression, suicide,depressive behavior response to tryptophan depletion, bipolar disorderantidepressant-induced mania and lesser anti-depressant response; and(3) association of a variant in HTR2C (serotonin receptor 2C) with bothrecurrent major depression and bipolar disorder and with majordepression.

Thereupon, as evidenced by the above, there is a need in the art toidentify proteins and genes associated with the pathophysiology ofdepression that are proteins and genes that relate to other thanserotonin or norepinephrine. Such proteins and genes would be useful inthe diagnosis of depression or a related disorder, and in thedevelopment of new drugs that could be used to treat patients sufferingfrom depression or a related disorder.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to an isolated nucleicacid molecule or fragment thereof comprising a nucleotide sequencehaving at least 90% identity to: (i) SEQ ID NO:2, (ii) nucleotides 352to 771 of SEQ ID NO:2; or (iii) nucleotides 812 to 1162 of SEQ ID NO:2or (iv) a complement comprising a nucleotide sequence having at least90% identity to: (i) SEQ ID NO:2, (ii) nucleotides 352 to 771 of SEQ IDNO:2; or (iii) nucleotides 812 to 1162 of SEQ ID NO:2. The presentinvention also encompasses a purified or isolated protein encoded by theabove nucleic acid molecule or fragment thereof.

In another embodiment, the present invention relates to a purifiedpolypeptide or fragment thereof comprising an amino acid sequence havingat least 90% identity to: SEQ ID NO:3 or SEQ ID NO:4.

In yet another embodiment, the present invention relates to a vectorcomprising:

a) an isolated nucleic acid sequence comprising a nucleotide sequencehaving at least 90% identity to: (i) SEQ ID NO:2, (ii) nucleotides 352to 771 of SEQ ID NO:2; or (iii) nucleotides 812 to 1162 of SEQ ID NO:2or a complement comprising a nucleotide sequence having at least 90%identity to: (i) SEQ ID NO:2, (ii) nucleotides 352 to 771 of SEQ IDNO:2; or (iii) nucleotides 812 to 1162 of SEQ ID NO:2; operably linkedto

b) a regulatory sequence.

Additionally, in yet another embodiment, the present invention relatesto a host cell comprising the above-described vector.

In still yet another embodiment, the present invention relates to anon-human transgenic animal. In one aspect, said non-human transgenicanimal comprises:

a) an exogenous and stably transmitted nucleic acid comprising anucleotide sequence of SEQ ID NO:2 (or any one or more of the sequencesdescribed above); or

b) a knock-out of a nucleic acid comprising a nucleotide sequence of SEQID NO:2 (or any one or more of the sequences described above).

In another aspect, said non-human transgenic animal comprises:

a) an exogenous and stably transmitted nucleic acid having a nucleotidesequence selected from the group consisting of: SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:31 and SEQ ID NO:33, with the proviso that said animaldoes not comprise an exogenous and stably transmitted nucleic acidhaving a nucleotide sequence of SEQ ID NO:2; or

b) a knock-out of a nucleic acid comprising a nucleotide sequenceselected from the group consisting of: SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO: 22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:31 and SEQ ID NO:33, with the proviso that a nucleic acid having anucleotide sequence of SEQ ID NO:2 is not knocked out.

In yet another embodiment, the present invention relates to a method ofmodifying or altering the expression of SEQ ID NO:2 in a cell or animal.The method involves the steps of:

a) exposing said cell or administering to said subject a nucleic acidmolecule, wherein said nucleic acid molecule modifies or alters theexpression of SEQ ID NO: 2; and

b) modifying or altering the expression of SEQ ID NO:2.

In the above-described method, the nucleic acid molecule can be anantisense molecule, a small interfering RNA, a co-suppression RNA, anaptamer, a ribozyme or a triplexing agent.

In yet another embodiment, the present invention relates to a method ofmodifying or altering the expression of a nucleic acid sequence having anucleotide sequence selected from the group consisting of: SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:33 in a cell or animal, withthe proviso that the expression of a nucleic acid having the sequence ofSEQ ID NO: 2 is not modified or altered. The method involves the stepsof:

a) exposing said cell or administering to said subject a nucleic acidmolecule, wherein said nucleic acid molecule modifies or alters theexpression of a nucleotide sequence selected from the group consistingof: SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:26,SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:33; and

b) modifying or altering the expression of SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,SEQ ID NO:31 or SEQ ID NO:33.

In the above-described method, the nucleic acid molecule can be anantisense molecule, a small interfering RNA, a co-suppression RNA, anaptamer, a ribozyme or a triplexing agent.

In yet another embodiment, the present invention relates to a method ofdetermining a genotype of a subject at a polymorphic site in nucleotides1 to 316 of SEQ ID NO: 2 in a test sample. The method involves the stepsof:

a) obtaining a test sample comprising DNA of a subject;

b) analyzing the test sample for at least one polymorphic site innucleotides 1 to 316 of SEQ ID NO:2;

c) identifying the allele(s) present at said polymorphic site in saidtest sample; and

d) determining the genotype of a subject based on the identification ofthe allele(s) at said polymorphic site in said test sample.

The above-described method can further involve the step of analyzing thetest sample(s) for a C-G polymorphism at position −1019 in a humanserotonin receptor 1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using direct sequencing, polymerase chain reaction, ligasechain reaction, a fragment length polymorphism assay, a single strandconformation polymorphism analysis, a heteroduplex assay, hybridization,Taqman®, Molecular Beacon, Pyrosequencing, a microarray, Southernblotting, an Invader assay, a single base extension assay, or massspectrometry.

In still yet another embodiment, the present invention relates to amethod of determining a genotype of a subject at nucleotides 77402 or79906 of SEQ ID NO: 1 in a test sample. The method involves the stepsof:

a) obtaining a test sample comprising DNA of a subject;

b) analyzing the test sample for at least one polymorphic site selectedfrom the group consisting of nucleotides 77402 and 79906 of SEQ ID NO:1;

c) determining the allele(s) present at said polymorphic site in saidtest sample; and

d) determining the genotype of a subject based on the identification ofthe allele(s) at said polymorphic site in said test sample.

The above-described method can further involve the step of analyzing thetest sample(s) for a C-G polymorphism at position −1019 in a humanserotonin receptor 1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using direct sequencing, polymerase chain reaction, ligasechain reaction, a fragment length polymorphism assay, a single strandconformation polymorphism analysis, a heteroduplex assay, hybridization,Taqman®, Molecular Beacon, Pyrosequencing, a microarray, Southernblotting, an Invader assay, a single base extension assay, or massspectrometry.

In still yet another embodiment, the present invention relates to amethod of identifying a subject having major depression or a relateddisorder, or at risk of developing major depression or a relateddisorder. The method involves the steps of:

a) obtaining a test sample subject comprising DNA of a subject;

b) analyzing the test sample for at least one polymorphic site in SEQ IDNO: 1;

c) identifying at least one allele at said polymorphic site; and

d) identifying whether said subject has major depression or a relateddisorder or is at risk of developing major depression or a relateddisorder based on the allele(s) identified at said polymorphic site(s)in said test sample.

The above-described method can further involve the step of analyzing thetest sample(s) for a C-G polymorphism at position −1019 in a humanserotonin receptor 1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using direct sequencing, polymerase chain reaction, ligasechain reaction, a fragment length polymorphism assay, a single strandconformation polymorphism analysis, a heteroduplex assay, hybridization,Taqman®, Molecular Beacon, Pyrosequencing, a microarray, Southernblotting, an Invader assay, a single base extension assay, or massspectrometry.

In still yet another embodiment, the present invention relates to amethod of providing a prognosis for or predicting a response totreatment for a subject having major depression or a related disorder.The method involves the steps of:

a) obtaining a test sample comprising DNA of a subject;

b) analyzing the test sample for at least one polymorphic site in SEQ IDNO: 1;

c) identifying at least one allele(s) at said polymorphic site; and

d) providing a prognosis for and predicting the response to treatmentfor a subject having major depression or a related disorder based on theallele(s) identified at said polymorphic site(s) in said test sample.

The above-described method can further involve the step of analyzing thetest sample(s) for a C-G polymorphism at position −1019 in a humanserotonin receptor 1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using direct sequencing, polymerase chain reaction, ligasechain reaction, a fragment length polymorphism assay, a single strandconformation polymorphism analysis, a heteroduplex assay, hybridization,Taqman®, Molecular Beacon, Pyrosequencing, a microarray, Southernblotting, an Invader assay, a single base extension assay, or massspectrometry.

In still yet another embodiment, the present invention relates to amethod of detecting or quantifying an mRNA which comprises nucleotides 1to 316 of SEQ ID NO:2 in a test sample. The method involves the stepsof:

a) obtaining a test sample subject comprising mRNA of a subject;

b) analyzing the test sample for a mRNA comprising at least 15contiguous nucleotides of nucleotides 1 to 316 of SEQ ID NO:2; and

b) detecting or quantifying said mRNA in said test sample.

The above-described method can further involve the step of analyzing thetest sample(s) for an mRNA transcribed from a human serotonin receptor1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using reverse transcription, quantitative polymerase chainreaction, cDNA microarrays, or Northern blotting.

In still yet another embodiment, the present invention relates to amethod of identifying a subject having major depression or a relateddisorder, or at risk of developing major depression or a relateddisorder. The method involves the steps of:

a) obtaining a test sample subject comprising subject mRNA;

b) analyzing the test sample for at least one mRNA transcribed from SEQID NO: 1; and

c) identifying whether said subject has major depression or a relateddisorder or is at risk of developing major depression or a relateddisorder based on the presence, absence or amount of at least one of themRNAs recited in step b) in said test sample.

The above-described method can further involve the step of analyzing thetest sample(s) for an mRNA transcribed from a human serotonin receptor1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using reverse transcription, quantitative polymerase chainreaction, cDNA microarrays, or Northern blotting.

In the above-described method, the mRNA transcribed from SEQ ID NO: 1can have the nucleotide sequence of: SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 orSEQ ID NO:33.

In still yet another embodiment, the present invention relates to amethod of providing a prognosis for or predicting a response totreatment for a subject having major depression or a related disorder.The method involves the steps of:

a) obtaining a test sample comprising mRNA of a subject;

b) analyzing the test sample for at least one mRNA transcribed from SEQID NO: 1; and

c) providing a prognosis for and predicting the response to treatmentfor a subject having major depression or a related disorder based on thepresence, absence or amount of at least one of the mRNAs recited in stepb) in said test sample.

The above-described method can further involve the step of analyzing thetest sample(s) for an mRNA transcribed from a human serotonin receptor1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using reverse transcription, quantitative polymerase chainreaction, cDNA microarrays, or Northern blotting.

In the above-described method, the mRNA transcribed from SEQ ID NO: 1can have the nucleotide sequence of: SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 orSEQ ID NO:33.

In still yet a further embodiment, the present invention relates to amethod of detecting or quantifying the amount of a protein having anamino acid sequence selected from the group consisting of: SEQ ID NO:3and SEQ ID NO:4 in a test sample. The method involves the steps of:

a) obtaining a test sample subject comprising at least one polypeptideof a subject; and

b) detecting or quantifying the amount of a protein having an amino acidsequence selected from the group consisting of SEQ ID NO: 3 and SEQ IDNO:4 in said test sample.

The above-described method can further involve the step of detecting orquantifying the amount of a polypeptide encoded by a human serotoninreceptor 1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using ELISA, RIA, Western blotting, fluorescence activatedcell sorting or immunohistochemical analysis.

In still yet a further embodiment, the present invention relates to amethod of identifying a subject having major depression or a relateddisorder, or at risk of developing major depression or a relateddisorder. The method involves the steps of:

a) obtaining a test sample comprising at least one polypeptide of asubject;

b) analyzing the test sample for at least one polypeptide translatedfrom SEQ ID NO:1; and

c) identifying whether said subject has major depression or a relateddisorder or is at risk of developing major depression or a relateddisorder based on the presence, absence or amount of at least of thepolypeptides recited in step b) in said test sample.

The above-described method can further involve the step of analyzing thetest sample(s) for a polypeptide encoded by a human serotonin receptor1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using ELISA, RIA, Western blotting, fluorescence activatedcell sorting or immunohistochemical analysis.

In the above-described method, the polypeptide translated from SEQ IDNO:1 can have an amino acid sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet a further embodiment, the present invention relates to amethod of providing a prognosis for or predicting a response totreatment for a subject having major depression or a related disorder.The method involves the steps of:

a) obtaining a test sample comprising at least one polypeptide of asubject;

b) analyzing the test sample for at least one polypeptide translatedfrom SEQ ID NO:1; and

c) providing a prognosis for and predicting the response to treatmentfor a subject having major depression or a related disorder based on thepresence, absence or amount of at least one of the polypeptides recitedin step b) in said test sample.

The above-described method can further involve the step of analyzing thetest sample(s) for a polypeptide encoded by a human serotonin receptor1A (HTR1A) gene.

The analyzing performed in the above-described method can beaccomplished using ELISA, RIA, Western blotting, fluorescence activatedcell sorting or immunohistochemical analysis.

In the above-described method, the polypeptide translated from SEQ IDNO:1 can have an amino acid sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to a kit.In one aspect, the kit can comprise:

a) at least one reagent for determining a genotype of a subject at apolymorphic site in SEQ ID NO: 1 in a test sample; and

b) instructions for determining the genotype of the subject.

In another aspect, the kit can comprise:

a) at least one reagent for determining a genotype of a subject for aC-G polymorphism at position −1019 in a human serotonin receptor 1A(HTR1A) gene;

b) at least one reagent for determining a genotype of a subject for apolymorphic site in SEQ ID NO: 1, in a test sample; and

c) instructions for determining the genotype of the subject.

In another aspect, the kit can comprise:

a) at least one reagent for determining a genotype of a subject atnucleotide 77402 of SEQ ID NO:1 in a test sample; and

b) instructions for determining the genotype of the subject.

In another aspect, the kit can comprise:

a) at least one reagent for determining a genotype of a subject atnucleotide 79906 of SEQ ID NO:1 in a test sample; and

b) instructions for determining the genotype of the subject.

In another aspect, the kit can comprise:

a) at least one reagent for determining a genotype of a subject for aC-G polymorphism at position −1019 in a human serotonin receptor 1A(HTR1A) gene;

b) at least one reagent for determining a genotype of a subject for apolymorphic site at at least one of nucleotides 77402 or 79906 in SEQ IDNO:1, in a test sample; and

c) instructions for determining the genotype of the subject.

In still yet another aspect, the kit comprises:

a) at least one reagent for detecting or quantifying an mRNA transcribedfrom SEQ ID NO:1 in a test sample; and

b) instructions for detecting or quantifying the mRNA transcribed fromSEQ ID NO:1 in the test sample.

In still yet another aspect, the kit comprises:

a) at least one reagent for detecting or quantifying an mRNA from aserotonin receptor 1A (HTR1A) gene in a test sample;

b) at least one reagent for detecting or quantifying an mRNA transcribedfrom SEQ ID NO:1, in a test sample; and

c) instructions for detecting or quantifying the mRNA from a serotoninreceptor 1A (HTR1A) gene and the mRNA transcribed from SEQ ID NO:1 inthe test sample.

The mRNA transcribed from SEQ ID NO:1 in the above kits in can have thenucleotide sequence of: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ IDNO:33.

It still yet another aspect, the kit comprises:

a) at least one reagent for detecting or quantifying a polypeptidetranslated from SEQ ID NO:1 in a test sample; and

b) instructions for detecting or quantifying the polypeptide translatedfrom SEQ ID NO:1 in the test sample.

In still yet another aspect, the kit comprises:

a) at least one reagent for detecting or quantifying a polypeptideencoded by a serotonin receptor 1A (HTR1A) gene in a test sample;

b) at least one reagent for detecting or quantifying a polypeptidetranslated from SEQ ID NO:1, in a test sample; and

c) instructions for detecting or quantifying the polypeptide encoded bya serotonin receptor 1A (HTR1A) gene and the polypeptide translated fromSEQ ID NO:1 in the test sample.

The polypeptide translated from SEQ ID NO:1 in the above-described kitscan have an amino acid sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of screening a composition for the ability to bind to a proteintranslated from SEQ ID NO:1. The method involves the steps of:

a) exposing said protein to a composition for a time and underconditions sufficient for said test composition to bind to said proteinto form protein/composition complexes; and

b) detecting presence or absence of said complexes, wherein the presenceof said complexes indicates a composition having the ability to bind tosaid protein.

The presence or absences of the complexes in the above-described methodcan be detected using mass spectrometry. Additionally, a compositionidentified pursuant to the above-described method as having the abilityto bind to a protein translated from SEQ ID NO:1 can be used to treatmajor depression or a related disorder in a subject.

In the above-described method, a protein translated from SEQ ID NO:1 canhave an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of detecting binding of a composition to a protein translatedfrom SEQ ID NO:1. The method involves the steps of:

a) subjecting said protein to nuclear magnetic resonance and recordingthe resulting spectrum;

b) subjecting said protein to nuclear magnetic resonance in the presenceof said composition and recording the resulting spectrum; and

c) detecting the difference between said spectrum of step a) and saidspectrum of step b) and comparing said difference to a control, saidcomparison indicating whether said composition binds to said protein.

A composition identified pursuant to the above-described method ashaving the ability to bind to a protein translated from SEQ ID NO:1 canbe used to treat major depression or a related disorder in a subject.

In the above-described method, a protein translated from SEQ ID NO:1 canhave an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of identifying the structure of a composition bound to a proteintranslated from SEQ ID NO:1. The method involves the steps of:

a) exposing said protein to a composition known to bind to said protein;and

b) observing the resulting X-ray diffraction pattern of said resultingbound composition of step a), said diffraction pattern indicating thestructure of said composition.

Additionally, a composition identified pursuant to the above-describedmethod as having the ability to bind to a protein translated from SEQ IDNO:1 can be used to treat major depression or a related disorder in asubject.

In the above-described method, a protein translated from SEQ ID NO:1 canhave an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of screening a composition for the ability to modulate theactivity of a protein translated from SEQ ID NO:1. The method involvesthe steps of:

a) providing a composition;

b) exposing the protein to a substrate for sufficient time andconditions to allow the substrate to react with the protein in order toproduce a reaction product or complex;

c) exposing the protein to the composition; and

d) measuring said reaction product or complex, wherein a decreased orincreased amount of said reaction product or complex, as compared to theamount of reaction product or complex produced in the absence of saidcomposition, indicates a composition having the ability to modulate theactivity of said protein.

In the above-described method, a protein translated from SEQ ID NO:1having an amino acid sequence selected from the group consisting of SEQID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:27 may be capable ofmodifying the phosphorylation of the substrate. Additionally, thesubstrate can be selected from the group consisting of:phosphohistidine, phospholysine, phosphodiimide, pyrophosphate and apeptide or protein phosphorylated on histidine or lysine.

Furthermore, a composition identified in the above-identified method ashaving an ability to modulate the activity of a protein can be used totreat major depression or a related disorder in a subject.

Moreover, in the above-described method, a protein translated from SEQID NO:1 can have an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of screening a composition for the ability to modulate theactivity of a protein translated from SEQ ID NO:1. The method involvesthe steps of:

a) providing a composition;

b) simultaneously exposing the protein to the composition and to asubstrate, wherein the protein is exposed to the substrate forsufficient time and conditions to allow the substrate to react with theprotein in order to produce a reaction product or complex; and

c) measuring presence or absence of said reaction product or complex,wherein a lack of said reaction product or complex indicating acomposition having the ability to modulate the activity of said protein.

In the above-described method, a protein translated from SEQ ID NO:1having an amino acid sequence selected from the group consisting of SEQID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:27 may be capable ofmodifying the phosphorylation of the substrate. Additionally, thesubstrate can be selected from the group consisting of:phosphohistidine, phospholysine, phosphodiimide, pyrophosphate and apeptide or protein phosphorylated on histidine or lysine.

Furthermore, a composition identified in the above-identified method ashaving an ability to modulate the activity of a protein can be used totreat major depression or a related disorder in a subject.

Moreover, in the above-described method, a protein translated from SEQID NO:1 can have an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of screening a composition for the ability to modulate activityof a protein translated from SEQ ID NO:1, in a cell. The method involvesthe steps of:

a) exposing said cell to said composition; and

b) measuring the amount of activity of said protein in said cell,wherein a decreased or increased amount of activity of said protein, ascompared to a cell which has not been exposed to said composition,indicates a composition having the ability to modulate the activity ofsaid protein.

A composition identified in the above-identified method as having anability to modulate the activity of a protein can be used to treat majordepression or a related disorder in a subject.

In the above-described method, a protein translated from SEQ ID NO:1 canhave an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of screening a composition for the ability to modulate expressionof a protein translated from SEQ ID NO:1, in a cell. The method involvesthe steps of:

a) exposing said cell to said composition; and

b) measuring the amount of said protein in said cell, wherein adecreased or increased amount of said protein, as compared to a cellwhich has not been exposed to said composition, indicates a compositionhaving the ability to modulate the expression of said protein.

A composition identified pursuant to the above-described method ashaving an ability to modulate the expression of the protein can be usedto treat major depression or a related disorder in a subject.

In the above-described method, a protein translated from SEQ ID NO:1 canhave an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of screening a composition for the ability to modulate the levelof expression of a protein translated from SEQ ID NO:1. The methodcomprises the steps of:

a) exposing an in vitro transcription and translation system comprisinga regulatory sequence from SEQ ID NO:1 functionally connected to theopen reading frame for a detectable protein, to a composition for a timeand under conditions sufficient for said test whether said compositionmodulates the level of expression of the detectable protein; and

b) detecting the level of expression of the detectable protein, whereina reduction or an increase in the level of expression of the detectableprotein indicates that said composition has the ability to modulate thelevel of expression of a protein translated from SEQ ID NO:1.

A composition identified pursuant to the above-described method ashaving an ability to modulate the level of expression of the protein canbe used to treat major depression or a related disorder in a subject.

In the above-described method, a protein translated from SEQ ID NO:1 canhave an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of treating major depression or a related disorder in a subjectin need of said treatment comprising the step of administering acomposition identified as modulating the activity of a proteintranslated from SEQ ID NO:1, to said subject, in an amount sufficient toeffect said treatment.

The composition administered to a subject pursuant to theabove-described method can: (a) inhibit or reduce the activity of theprotein; (b) increase the activity of the protein; or (c) decrease theactivity of the protein.

A protein translated from SEQ ID NO:1 and that can be used in theabove-described method can have an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ IDNO:34.

In still yet another embodiment, the present invention relates to amethod of treating major depression or a related disorder in a subjectin need of said treatment comprising reducing the amount of a proteintranslated from SEQ ID NO:1 in said subject, to a level sufficient toeffect said treatment.

In the above-described method, the reduction can result from completebinding or partial binding of a composition to said protein. A proteintranslated from SEQ ID NO:1 and that can be used in the above-describedmethod can have an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of treating major depression or a related disorder in a subjectin need of said treatment comprising increasing the amount of a proteintranslated from SEQ ID NO:1, to a level sufficient to effect saidtreatment.

In addition, the above-described method can involve administering tosaid subject a therapeutically effective amount of a protein translatedfrom SEQ ID NO:1. A protein translated from SEQ ID NO:1 and that can beused in the above-described method can have an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of treating major depression or a related disorder in a subjectin need of said treatment comprising the step of administering acomposition identified as modulating the level of expression of an mRNAmolecule transcribed from SEQ ID NO: 1 to said subject, in an amountsufficient to effect said treatment.

In the above-described method, an mRNA molecule transcribed from SEQ IDNO:1 can have the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:31 or SEQ ID NO:33.

In still yet another embodiment, the present invention relates to amethod of determining the therapeutic activity of a composition used totreat major depression or a related disorder. The method involves thesteps of:

a) determining the amount of a protein translated from SEQ ID NO:1, in atest sample from a subject treated with said composition; and

b) comparing the amount of said protein in said test sample with theamount of protein present in a test sample from said subject prior totreatment, an equal amount of said protein in said test sample of saidtreated subject indicating lack of therapeutic activity of saidcomposition and a changed amount of said protein in said test sample ofsaid treated subject indicating therapeutic activity of saidcomposition.

In the above-described method, a protein translated from SEQ ID NO:1 canhave an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of determining the level of therapeutic activity of a compositionused to treat major depression or a related disorder. The methodinvolves the steps of:

a) determining the activity of a protein translated from SEQ ID NO:1, ina test sample from a subject treated with said composition; and

b) comparing the amount of activity of said protein in said test samplewith the amount of activity of protein present in a test sample fromsaid subject prior to treatment, an equal amount of activity of saidprotein in said test sample of said treated subject indicating lack oftherapeutic activity of said composition and a changed amount ofactivity of said protein in said test sample of said treated subjectindicating therapeutic activity of said composition.

In the above-described method, a protein translated from SEQ ID NO:1 canhave an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of determining the level of in vivo activity of a compositionused to treat major depression or a related disorder comprising thesteps of:

a) determining the amount of a mRNA molecule transcribed from SEQ IDNO:1, in a test sample from a subject treated with said composition; and

b) comparing the amount of said mRNA molecule in said test sample withthe amount of mRNA molecule present in a test sample from said subjectprior to treatment, an equal amount of said mRNA molecule in said testsample of said treated subject indicating lack of therapeutic activityof said composition and a changed amount of said mRNA molecule in saidtest sample of said treated subject indicating therapeutic activity ofsaid composition.

In the above-described method, an mRNA molecule transcribed from SEQ IDNO:1 can have the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:31 or SEQ ID NO:33.

In still yet another embodiment, the present invention relates to amethod of determining presence or absence of activity of a compositionused to treat major depression or a related disorder. The methodinvolves the steps of:

a) observing phenotype of a subject according to a method validated as ameasure of major depression or a related disorder;

b) administering said composition to said subject for a time and underconditions sufficient for said composition to bind to, inhibit, increaseor reduce the activity of, or increase or reduce the amount of a proteintranslated from SEQ ID NO:1;

c) repeating step a) with said subject of step b); and

d) comparing said phenotype of step a) and said phenotype of step c), adifference in step c) as compared to step a) indicating presence ofactivity of said composition and the lack of a difference indicatingabsence of activity of said composition.

In the above-described method, a protein translated from SEQ ID NO:1 canhave an amino acid sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

In still yet another embodiment, the present invention relates to amethod of determining presence or absence of activity of a compositionused to treat major depression or a related disorder comprising thesteps of:

a) observing phenotype of a subject according to a method validated as ameasure of major depression or a related disorder;

b) administering said composition to said subject for a time and underconditions sufficient for said composition to increase or reduce theamount of a mRNA molecule transcribed from SEQ ID NO:1;

c) repeating step a) with said subject of step b); and

d) comparing said phenotype of step a) and said phenotype of step c), adifference in step c) as compared to step a) indicating presence ofactivity of said composition and the lack of a difference indicatingabsence of activity of said composition.

In the above-described method, an mRNA molecule transcribed from SEQ IDNO:1 can have the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:31 or SEQ ID NO:33.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic DNA sequence comprising DEP2 (SEQ ID NO:1). Thecapital letters show the open reading frame for human phospholysinephosphohistidine inorganic pyrophosphate phosphatase (Lhpp) protein. Redtext indicates single nucleotide polymorphisms. The bold underlinedportions are forward primers. The bold italic underlined portions arereverse primers. The italic portions are repeat regions. The beginningportion of the sequence before the gap is disclosed as SEQ ID NO: 70.

FIG. 2 shows the nucleic acid sequence of DEP2-1 (SEQ ID NO:2). Thenucleic acid sequence contains two (2) coding regions, each of which areshown in capital letters.

FIG. 3A shows the amino acid sequence of the Dep2-1a protein encoded byone of the coding regions of the nucleic acid shown in FIG. 2 (SEQ IDNO:3).

FIG. 3B shows the amino acid sequence of the Dep2-1b protein encoded bythe second coding region of the nucleic acid shown in FIG. 2 (SEQ IDNO:4).

FIG. 4 shows the nucleic acid sequence of a naturally occurring splicevariant of DEP2-1 (SEQ ID NO:5). The coding regions are shown in capitalletters. The amino acid sequences of the proteins encoded by each of thecoding regions is the same as shown in SEQ ID NOS: 3 and 4.

FIG. 5 shows the nucleic acid sequence of a naturally occurring splicevariant of DEP2-1 (SEQ ID NO:6). The coding regions are shown in capitalletters. The amino acid sequences of the proteins encoded by each of thecoding regions is the same as shown in SEQ ID NOS: 3 and 4.

FIG. 6 shows the nucleic acid sequence of a naturally occurring splicevariant of DEP2-1 (SEQ ID NO:7). The coding region is shown in capitalletters. The amino acid sequence of the protein encoded by the codingregion is the same as shown in SEQ ID NO:4.

FIG. 7 shows the nucleic acid sequence of a naturally occurring splicevariant of DEP2-1 (SEQ ID NO:8). The coding region is shown in capitalletters. The amino acid sequence of the protein encoded by the codingregion is the same as shown in SEQ ID NO:4.

FIG. 8 shows the reference nucleic acid sequence of LHPP (SEQ ID NO:9).The coding region is shown in capital letters.

FIG. 9 shows the amino acid sequence of the Lhpp protein encoded by thenucleic acid shown in FIG. 2 (SEQ ID NO:10). A polymorphic amino acidhas been found to exist at amino acid 94 where arginine is replaced byglutamine (see the underline).

FIG. 10 shows a naturally occurring splice variant of LHPP (SEQ IDNO:11). The coding region is shown in capital letters. The coding regionencodes a protein that is identical to SEQ ID NO:10.

FIG. 11 shows a naturally occurring splice variant of LHPP (SEQ IDNO:12). The coding region is shown in capital letters.

FIG. 12 shows the amino acid sequence of the variant Lhpp proteinencoded by the nucleic acid shown in FIG. 5 (SEQ ID NO:13).

FIG. 13 shows a naturally occurring splice variant of LHPP (SEQ IDNO:14). The coding region is shown in capital letters. The amino acidsequence of the variant Lhpp protein encoded by the nucleic acid isshown in SEQ ID NO:15.

FIG. 14 shows a nucleic acid sequence of a naturally occurring splicevariant of LHPP (SEQ ID NO:16). The coding region is shown in capitalletters. The amino acid sequence of the variant Lhpp protein encoded bythe nucleic acid is shown in SEQ ID NO: 17.

FIG. 15A and FIG. 15B show a nucleic acid sequence of a naturallyoccurring splice variant of LHPP (SEQ ID NO:18). The coding region isshown in capital letters. The amino acid sequence of the variant Lhppprotein encoded by the nucleic acid is shown in SEQ ID NO:19.

FIG. 16 shows a nucleic acid of a naturally occurring splice variant ofLHPP (SEQ ID NO:20). The coding region is shown in capital letters. Theamino acid sequence of the variant Lhpp protein encoded by the nucleicacid is shown in SEQ ID NO:21.

FIG. 17 shows a nucleic acid of a naturally occurring splice variant ofLHPP (SEQ ID NO:22). The coding region is shown in capital letters. Theamino acid sequence of the variant Lhpp protein encoded by the nucleicacid is shown in SEQ ID NO:23.

FIG. 18 shows a nucleic acid of a naturally occurring splice variant ofLHPP (SEQ ID NO:24). The coding region is shown in capital letters. Theamino acid sequence of the variant Lhpp protein encoded by the nucleicacid is shown in SEQ ID NO:25.

FIG. 19 shows a nucleic acid of a naturally occurring splice variant ofLHPP (SEQ ID NO:28). The coding region is shown in capital letters. Theamino acid sequence of the protein encoded by the nucleic acid is shownin SEQ ID NO:29.

FIG. 20 shows a nucleic acid sequence of DEP2-2 (SEQ ID NO:26). Thecoding region is shown in capital letters.

FIG. 21 shows the amino acid sequence of the Dep2-2 protein encoded bythe nucleic acid shown in FIG. 20 (SEQ ID NO:27).

FIG. 22 shows a nucleic acid sequence of DEP2-3 (SEQ ID NO:30).

FIG. 23A and FIG. 23B show a nucleic acid of AK127935 (GenBank:AK127935) (SEQ ID NO:31). The coding region is shown in capital letters.

FIG. 24 shows the amino acid sequence of the Dep2-4 protein encoded bythe nucleic acid shown in FIG. 23 (SEQ ID NO:32).

FIG. 25A and FIG. 25B show a nucleic acid of AW867792 (GenBank:AW867792) (SEQ ID NO:33). The coding region is shown in capital letters.

FIG. 26 shows the amino acid sequence of the Dep2-5 protein encoded bythe nucleic acid shown in FIG. 25 (SEQ ID NO:34).

FIG. 27 shows the genetic evidence on chromosome 10 for linkage of agene to major depressive disorder. The lower, curved line and lower, twohorizontal lines show evidence from standard linkage analysis. The greenlines show evidence from serotonin receptor 1A-conditional linkageanalysis. The dotted horizontal lines indicate the extent of the linkageregion as defined by a drop of one unit in the heterogeneity LOD score.The dashed horizontal lines indicate the extent of the linkage region asdefined by a drop of two units in the heterogeneity LOD score. The rightside of the figure represents the telomere of chromosome 10. Theapproximate location of DEP2 is represented by an arrowhead.

FIG. 28 shows a Northern blot probed with a polynucleotide complementaryto SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:11 and SEQ ID NO:12.

FIG. 29 is a schematic representation of certain naturally occurringDEP2 transcripts and proteins. This figure was created using Genecartasoftware (Compugen, Tel Aviv, Israel). Dark lines represent transcriptsand light lines represent coding regions. Transcripts shown include LHPPand naturally occurring variants thereof, DEP2-1 and naturally occurringvariants thereof, and DEP2-2. DEP2-3, for which all supportive evidencehas been generated, is not shown. AK127935 and AW867792, which share noexons in common with LHPP or DEP2-1, or naturally occurring variantsthereof, are not shown. Although SEQ ID NO:2, SEQ ID NO:5 and SEQ IDNO:6 each contain the coding regions for both Dep2-1a and Dep2-1b, onlythe 5′-most (Dep2-1a) is associated with those transcripts in thisfigure. The location of Dep2-1b is associated with SEQ ID NO:7 and SEQID NO:8.

FIG. 30 shows a sequence alignment of DEP2-1 sequences either predictedby a bioinformatic algorithm (Genecarta (SEQ ID NO:71)) or determinedexperimentally by direct sequencing of cloned cDNAs h5173309 (SEQ IDNO:72), h5194531 (SEQ ID NO:73), h3197955 (SEQ ID NO:74) and h4565014(SEQ ID NO:75). Arrowheads indicate major transcription start sitesdetermined by RLM-RACE. A single nucleotide polymorphism is indicated byan underlined base in the h4565014 sequence. The last line of sequencewas found, downstream of a polyadenylate tract, in h4565014 and does notmatch DEP2.

FIG. 31 shows RLM-RACE results. MWM=molecular weight markers.

FIG. 32 shows the results of exon bridging PCR experiment #1 in Example7. The lower band between 50 and 100 nucleotide markers are primerdimers.

FIG. 33 shows the results of exon bridging PCR experiment #2 in Example7. Negative controls are reactions in which no reverse transcriptase wasadded.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the singular forms “a,” “an” and “the” include pluralreference unless the context clearly dictates otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood to one those of skill in the art to whichthis invention belongs.

As used herein, the term “allele” refers to a particular form of anucleic acid, either DNA or RNA, wherein different alleles of a nucleicacid differ in sequence, by either change or insertion/deletion, at oneor more nucleotides at a polymorphic site.

“cDNA” refers to a DNA that is complementary to and synthesized from amRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form.

As used herein, the term “coding sequence” or “coding region” refers toa nucleic acid sequence that codes for a specific amino acid sequence.

As used herein, the term “complementarity” refers to as the degree ofrelatedness between two nucleic acid segments. It is determined bymeasuring the ability of the sense strand of one nucleic acid segment tohybridize with the antisense strand of the other nucleic acid segment,under appropriate conditions, to form a double helix. A “complement” isdefined as a sequence which pairs to a given sequence based upon thecanonic base-pairing rules. For example, a sequence A-G-T in onenucleotide strand is “complementary” to T-C-A in the other strand.

In the DNA double helix, wherever adenine appears in one strand, thymine(uridine in RNA) appears in the other strand. Similarly, whereverguanine is found in one strand, cytosine is found in the other. Thegreater the relatedness between the nucleotide sequences of two nucleicacid segments, the greater the ability to form hybrid duplexes betweenthe strands of the two nucleic acid segments.

“Similarity” between two amino acid sequences is defined as the presenceof a series of identical as well as conserved amino acid residues inboth sequences. The higher the degree of similarity between two aminoacid sequences, the higher the correspondence, sameness or equivalenceof the two sequences. (“Identity” between two amino acid sequences isdefined as the presence of a series of exactly alike or invariant aminoacid residues in both sequences.) The definitions of “complementarity”,“identity” and “similarity” are well known to those of ordinary skill inthe art.

As used herein, the term “DEP2” refers to a gene on human chromosome10q26.2 that has been statistically linked and associated with majordepression, and that is believed to be within the 159 kb sequencecomprising SEQ ID NO:1. Transcripts that arise from DEP2 include: (a)LHPP (SEQ ID NO:9) (See, Yokoi et al., J Biochem 133:607-14 (2003)); (b)naturally occurring splice variants of LHPP (SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24 and SEQ ID NO:26; (c) DEP2-1 (SEQ ID NO:2); (d) naturallyoccurring splice variants of DEP2-1 (SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7 or SEQ ID NO:8); (e) DEP2-2 (SEQ ID NO:28); (f) DEP2-3 (SEQ IDNO:30); (g) GenBank sequence AK127935 (SEQ ID NO:31); and (h) GenBanksequence AW867792 (SEQ ID NO:33). Proteins that are encoded within DEP2include: (a) Lhpp (SEQ ID NO:10) (See, Yokoi et al., J Biochem133:607-14 (2003)); (b) naturally occurring protein variants of Lhpp(SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:27); (c) Dep2-1a and Dep2-1b(SEQ ID NO:3 and SEQ ID NO:4, respectively); (d) Dep2-2 (SEQ ID NO:29);(e) Dep2-4 (SEQ ID NO:32); and (f) Dep2-5 (SEQ ID NO:34).

As used herein, the term “DEP2 transcripts” refers to the group oftranscripts arising in whole or in part from SEQ ID NO:1, including butnot limited to: LHPP and naturally occurring splice variants thereof;DEP2-1 and naturally occurring splice variants thereof; DEP2-2; DEP2-3;GenBank sequence AK127935 and GenBank sequence AW867792. As used herein,the term “DEP2 proteins” refers to the group of proteins encoded inwhole or in part from one or more DEP2 transcripts, including but notlimited to: Lhpp and naturally occurring protein variants thereof;Dep2-1a; Dep2-1b; Dep2-2; Dep2-4 and Dep2-5. As used herein, the terms“DEP2 polymorphic sites” or “DEP2 polymorphisms”, used interchangeably,refer to polymorphic sites found within SEQ ID NO:1, or if outside ofSEQ ID NO:1, within a DEP2 transcript.

As used herein, the term “DEP2-1” refers to a messenger RNA shown in SEQID NO:2 and in FIG. 2, and DNA sequences that functionally regulateexpression thereof. Experimental evidence that DEP2-1 messenger RNA is anaturally occurring transcript is disclosed herein. As shown in FIG. 2,DEP2-1 messenger RNA has 2 exons. Of these, an exon comprisingnucleotides 1-315 of SEQ ID NO:2 was not previously known to be in anynaturally occurring transcript. In addition, three naturally occurringpolymorphic sites in nucleotides 1-315 of DEP2-1 messenger RNA aredisclosed herein: (a) 135T>C, (b) 209A>G and (c) 241G>A.

As used herein, the terms “Dep2-1a” and “Dep2-1b” refer to proteinsshown in SEQ ID NO:3 and in FIG. 3A, and in SEQ ID NO:4 and in FIG. 3B,respectively. These proteins may be encoded from DEP2-1 as well asnaturally occurring splice variants thereof.

As used herein, the term “DEP2-2” refers to a messenger RNA shown in SEQID NO:28, and DNA sequences that functionally regulate expressionthereof.

As used herein, the term “Dep2-2” refers to a protein shown in SEQ IDNO:29. This protein may be encoded from DEP2-2.

As used herein, the term “DEP2-3” refers to a messenger RNA shown in SEQID NO:30, and DNA sequences that functionally regulate expressionthereof.

As used herein, the term “Dep2-4” refers to a protein shown in SEQ IDNO:32. This protein may be encoded from SEQ ID NO:31.

As used herein, the term “Dep2-5” refers to a protein shown in SEQ IDNO:34. This protein may be encoded from SEQ ID NO:33.

As used herein, the phrase “effective amount” or a “therapeuticallyeffective amount”, which are used interchangeably herein, when used inconnection with an active agent (such as a drug) is meant a nontoxic butsufficient amount of the active agent to provide the desired effect. Theamount of active agent (such as a drug) that is “effective” will varyfrom subject to subject, depending on the age and general condition ofthe individual, the particular active agent or agents, and the like.Thus, it is not always possible to specify an exact “effective amount.”However, an appropriate “effective amount” in any individual case may bedetermined by one of ordinary skill in the art using routineexperimentation.

As used herein, the phrase “encoded by” refers to a nucleic acidsequence which codes for a polypeptide sequence, wherein the polypeptidesequence or a portion thereof contains an amino acid sequence of atleast 3 amino acids, more preferably at least 8 amino acids, and evenmore preferably at least 15 amino acids from a polypeptide encoded bythe nucleic acid sequence.

As used herein, the term “exon” refers to a portion of the gene sequencethat is transcribed and is found in the mature messenger RNA derivedfrom the gene, but is not necessarily a part of the sequence thatencodes the final gene product.

The term “expression”, as used herein, refers to the production of afunctional end-product. Expression of a gene involves transcription ofthe gene and translation of the mRNA into a precursor or mature protein.“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the targettranscript. “Co-suppression” refers to the production of sense RNAtranscripts capable of suppressing the expression of identical orsubstantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020).

As used herein, the term “fragment” of a nucleic acid sequence refers toa contiguous sequence of approximately at least 6 nucleotides,preferably at least about 8 nucleotides, more preferably at least about10 nucleotides, and even more preferably at least about 15 nucleotides,and most preferable at least about 20 nucleotides identical orcomplementary to a region of the specified nucleotide sequence.)Nucleotides (usually found in their 5′-monophosphate form) are referredto by their single letter designation as follows: “A” for adenylate ordeoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, and “N” for any nucleotide.

As used herein, the term “gene” refers to a nucleic acid sequence thatundergoes transcription as a result of the activity of at least onepromoter. A gene may encode for a particular polypeptide, oralternatively, code for a RNA molecule. A gene includes one or moreexons and one or more regulatory or control sequences and may includeone or more introns. The phrase “target gene” as used herein, refers toa nucleic acid sequence, such as, but not limited to, a nucleic acidsequence of interest that encodes a polypeptide of interest oralternatively, a RNA molecule of interest. The term “target gene” canalso refer to a gene to be identified or knocked-out according to themethods described herein.

As used herein, the term “genotype” refers to the identity of allelespresent in a subject or in a test sample.

As used herein, the term “genotyping” refers to the process ofdetermining the genotype of a subject.

As used herein, the terms “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably. They refer tonucleic acid or protein fragments wherein changes in one or morenucleotide bases or amino acids does not affect the ability of thenucleic acid or protein fragment to mediate gene expression or produce acertain phenotype. These terms also refer to modifications of thenucleic acid or protein fragments of the instant invention such asdeletion or insertion of one or more nucleotides or amino acids that donot substantially alter the functional properties of the resultingnucleic acid or protein fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the sequencesexemplified herein.

As used herein, the term “identity” refers to the relatedness of twosequences on a nucleotide-by-nucleotide basis over a particularcomparison window or segment. Thus, identity is defined as the degree ofsameness, correspondence or equivalence between the same strands (eithersense or antisense) of two DNA segments (or two amino acid sequences).

“Percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over a particular region, determining thenumber of positions at which the identical base or amino acid occurs inboth sequences in order to yield the number of matched positions,dividing the number of such positions by the total number of positionsin the segment being compared and multiplying the result by 100. Optimalalignment of sequences may be conducted by the algorithm of Smith &Waterman, Appl. Math., 2:482 (1981), by the algorithm of Needleman &Wunsch, J. Mol. Biol., 48:443 (1970), by the method of Pearson & Lipman,Proc. Natl. Acad. Sci., (USA) 85:2444 (1988) and by computer programswhich implement the relevant algorithms (for example, Clustal MacawPileup (which is publicly available on the Internet; Higgins et al.,CABIOS. 5L151-153 (1989)), FASTDB (Intelligenetics), BLAST (NationalCenter for Biomedical Information; Altschul et al., Nucleic AcidsResearch, 25:3389-3402 (1997)), PILEUP (Genetics Computer Group,Madison, Wis.) or GAP, BESTFIT, FASTA and TFASTA (Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, Madison, Wis.).(See U.S. Pat. No. 5,912,120.)

As used herein, the term “isoform” refers to a particular form of aprotein, wherein different isoforms of a protein differ in sequence, byeither change or insertion/deletion, or covalent modification at one ormore amino acids.

As used herein, the terms “isolated” or “purified”, usedinterchangeably, when used in connection with biological molecules suchas nucleic acids or proteins means that the molecule is substantiallyfree of other biological molecules such as nucleic acids, proteins,lipids, carbohydrates or other material such as cellular debris andgrowth media. Generally, the term “isolated” or “purified” are notintended to refer to a complete absence of such material or to absenceof water, buffers, or salts, unless they are present in amounts thatsubstantially interfere with the methods of the present invention.

As used herein, an “isolated nucleic acid fragment or sequence” is apolymer of nucleic acid (RNA or DNA) that is single- or double-stranded,optionally containing synthetic, non-natural or altered nucleotidebases. An isolated nucleic acid fragment in the form of a polymer of DNAmay be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA.

As used herein, the term “Lhpp” refers to an enzyme known asphospholysine phosphohistidine inorganic pyrophosphate phosphatase, andthe term “LHPP” refers to the corresponding messenger RNA, and DNAsequences that functionally regulate expression thereof. Lhpp wasoriginally purified from swine brain in 1957 (See, Seal et al., J BiolChem 228:193-9 (1957)), and subsequently has been purified from severaladditional mammalian sources (See, Felix et al., J Biochem 147:111-8(1975); Yoshida et al., Cancer Research 42:3256-31 (1982); Hachimori etal., J Biochem 93:257-64 (1983); Smirnova et al., Arch Biochem Biophys287:135-40 (1991); Hiraishi et al., Arch Biochem Biophys 341:153-9(1997)). The enzyme has been characterized in vitro as efficientlycatalyzing the hydrolysis of P—N bonds in phosphohistidine andphospholysine, and less efficiently catalyzing the hydrolysis of P—N orP—O bonds in imidodiphosphate and pyrophosphate, respectively. Lhpp maybe a protein histidine or lysine phosphoamidase, i.e., an enzyme thatmodifies the N-linked phosphorylation state of other proteins. The humanLHPP has been cloned. Functional human Lhpp enzyme has been purifiedfollowing heterologous expression in E. coli (See, Yokoi et al., JBiochem 133:607-14 (2003)). The nucleic acid sequence of LHPP messengerRNA is shown in SEQ ID NO:9 and in FIG. 8. The amino acid sequence ofLhpp is shown in SEQ ID NO:10 and in FIG. 9. As shown in FIG. 1, LHPPmessenger RNA has 7 exons. The locations of these exons are providedbelow in Table A.

TABLE A Exon Start in SEQ ID NO: 1 End in SEQ ID NO: 1 1 3001 3163 225305 25492 3 29588 29741 4 38127 38190 5 39202 39294 6 58346 58437 7154430 155313In addition, there is a naturally occurring polymorphic site in Lhpp(R94Q) in which amino acid 94 is either arginine or glutamine in the twonaturally occurring isoforms. In the corresponding naturally occurringpolymorphic site in LHPP messenger RNA (281G>A), base 281 of the openreading frame is either guanine or adenine in the two naturallyoccurring alleles. Further, Lhpp is encoded from a naturally occurringsplice variant of LHPP that is shown in SEQ ID NO:11 (See FIG. 10).

As used herein, the term “locus” refers to a location on a chromosome ofa nucleic acid molecule corresponding to a gene or a physical orphenotypic feature, where physical features include polymorphic sites.

As used herein, the term “major depression or a related disorder” refersto any Mood Disorder or Anxiety Disorder described in the Diagnostic andStatistical Manual (DSM-IV-TR, American Psychiatric Association, 2000).Mood Disorders include, but are not limited to, Depressive Disorders(DSM-IV-TR 296.2x, 296.3x, 300.4, 311), Bipolar Disorders (DSM-IV-TR296.0x, 296.40, 296.4x, 296.5x, 296.6x, 296.7, 296.89, 301.13, 296.80)and Mood Disorder Not Otherwise Specified (DSM-IV-TR 296.90). AnxietyDisorders include, but are not limited to, Panic Disorders (DSM-IV-TR300.01, 300.21), Phobic Disorders (DSM-IV-TR 300.29, 300.22, 300.23),Obsessive-Compulsive Disorder (DSM-IV-TR 300.3), Post-Traumatic StressDisorder (DSM-IV-TR 309.81), Acute Stress Disorder (DSM-IV-TR 308.3),Generalized Anxiety Disorder (DSM-IV-TR 300.02) and Anxiety Disorder NotOtherwise Specified (DSM-IV-TR 300.00). Extensive lists of symptoms anddiagnostic criteria for each of these disorders are found in theDSM-IV-TR sections cited above.

As used herein, the terms “modulates” “modulation” or “modulating” asused interchangeably herein, refer to both upregulation (for example,activation or stimulation (for example, by agonizing or potentiating))and downregulation (for example, inhibition or suppression (for example,by antagonizing, reducing, decreasing or inhibiting)).

As used herein, the term “naturally occurring” refers to a DNA molecule,a messenger RNA, a protein, an allele, an isoform, a polymorphic site, asplice variant or a protein variant, wherein the existence in nature ofsaid DNA molecule, messenger RNA, protein, allele, isoform, polymorphicsite, splice variant or protein variant is supported by either (a)direct experimental evidence or (b) algorithmic assembly from a databaseof nucleic acid or protein sequences. Alleles, isoforms, polymorphicsites, splice variants and protein variants might also be created byexperimental manipulation.

As used herein, the term “naturally occurring splice variant of DEP2-1”includes but is not limited to the sequences shown in SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

As used herein, the term “naturally occurring splice variant of LHPP”includes but is not limited to the sequences shown in SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24 and SEQ ID NO:26. As used herein, the term “naturally occurringprotein variant of Lhpp” includes but is not limited to the sequencesshown in SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:27.

As used herein, the phrase “3′ non-coding sequences” refer to mRNAsequences located downstream of a coding sequence.

As used herein, the term “non-human animal” includes all vertebrateanimals, except humans. It also includes an individual animal in allstages of development, including embryonic and fetal stages. A“transgenic animal” is any animal containing one or more cells bearinggenetic information altered or received, directly or indirectly, bydeliberate genetic manipulation at a subcellular level, such as bytargeted recombination or microinjection or infection with recombinantvirus.

Mice are often used for transgenic animal models because they are easyto house, relatively inexpensive, and easy to breed. However, othernon-human transgenic animals may also be made in accordance with thepresent invention such as, but not limited to, primates, mice, goat,sheep, rabbits, dogs, cows, cats, guinea pigs, rats, zebrafish andnematodes. Transgenic animals are those which carry a transgene, thatis, a cloned gene introduced and stably incorporated which is passed onto successive generations.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term includes double- and single-strandedDNA, as well as double- and single-stranded RNA. It also includesmodifications, such as methylation or capping and unmodified forms ofthe polynucleotide. The terms “polynucleotide,” “oligomer,”“oligonucleotide,” and “oligo” are used interchangeably herein.

As used herein, the phrase “operably linked” refers to the associationof nucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

Polymerase chain reaction (“PCR”) is a technique used to amplify DNAmillions of fold, by repeated replication of a template, in a shortperiod of time. (Mullis et al., Cold Spring Harbor Symp. Quant. Biol.51:263-273 (1986); Erlich et al., European Patent Application No.50,424; European Patent Application No. 84,796; European PatentApplication No. 258,017; European Patent Application No. 237,362;Mullis, European Patent Application No. 201,184; Mullis et al., U.S.Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki et al.,U.S. Pat. No. 4,683,194). The process utilizes sets of specific in vitrosynthesized oligonucleotides to prime DNA synthesis. The design of theprimers is dependent upon the sequences of DNA that are desired to beanalyzed. The technique is carried out through many cycles (usually20-50) of melting the template at high temperature, allowing the primersto anneal to complementary sequences within the template and thenreplicating the template with DNA polymerase.

As used herein, the term “polymorphic site” refers to a nucleic acidsequence comprising one or more consecutive nucleotides that differbetween alleles, or to a protein sequence comprising one or moreconsecutive amino acids that differ between isoforms.

As used herein, the term “polymorphism” refers to a sequence variationobserved in a subject at a polymorphic site. Polymorphisms includenucleotide or amino acid substitutions, insertions and deletions andmay, but need not, result in detectable differences in gene expressionor protein function.

The terms “polypeptide” and “protein” are used interchangeably hereinand indicate at least one molecular chain of amino acids linked throughcovalent and/or non-covalent bonds. The terms do not refer to a specificlength of the product. Thus peptides, oligopeptides and proteins areincluded within the definition of polypeptide. In addition, proteinfragments, analogs, mutated or variant proteins, fusion proteins and thelike are included within the meaning of polypeptide.

As used herein, the term “primer” refers to an oligonucleotide, whethernaturally occurring, such as in a purified restriction digest, orproduced synthetically, which is capable of acting as a point ofinitiation of synthesis when placed under conditions in which synthesisof a primer extension product that is complementary to a nucleic acidstrand is induced (such as in the presence of nucleotides and aninducing agent such as DNA polymerase and at a suitable temperature andpH). Primers can be single or double stranded. If double stranded, theprimer is first treated to separate its strands before being used toprepare extension products. The exact length of the primers will dependon many factors, including temperature, source of primer and the use ofthe method. Primers preferably have a length of at least 10 contiguousnucleotides. For example, primers can have a length of 10 contiguousnucleotides, 15 contiguous nucleotides, 20 contiguous nucleotides, 25contiguous nucleotides, etc.

As used herein, the term “probe” refers to an oligonucleotide, whethernaturally occurring, such as in a purified restriction digest, producedsynthetically, recombinantly or by polymerase chain reactionamplification which is capable of hybridizing to another oligonucleotideor nucleic acid of interest. A probe may be single-stranded ordouble-stranded. Probes can be labeled with a detectable label so as tomake said probe detectable in a detection system. The detectable labelused is not critical.

As used herein, the term “promoter” refers to a DNA sequence capable ofcontrolling the transcription of a RNA. Promoters may be derived intheir entirety from a native gene, or be composed of different elementsderived from different promoters found in nature, or even comprisesynthetic DNA segments.

As used herein, the term “protein variant” refers to a polypeptide thatis encoded from a splice variant, wherein two protein variants differ inthe inclusion/exclusion of one or more blocks of consecutive aminoacids.

The terms “recombinant construct”, “construct”, “expression construct”and “recombinant expression construct” are used interchangeably herein.These terms refer to a functional unit of genetic material that can beinserted into the genome of a cell or expressed in vitro using standardmethodology well known to one skilled in the art. Such construct may beitself or may be used in conjunction with a vector. If a vector is usedthen the choice of vector is dependent upon the method that will be usedto transform host plants as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments of the invention.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al.,Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, Western analysisof protein expression, or phenotypic analysis.

As used herein, the term “regulatory sequences” refers to a DNA or RNAsequence capable of controlling the expression of a RNA or protein.Regulatory sequences may include, but are not limited to, promoters,translation leader sequences, introns, and polyadenylation recognitionsequences.

As used herein, the phrase “RNA transcript” or “RNA molecule” as usedinterchangeable herein, refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. “Messenger RNA(mRNA)” refers to the RNA that is without introns and that can betranslated into protein by the cell.

As used herein, the phrase “sense RNA” refers to RNA molecule thatincludes the mRNA and can be translated into protein within a cell or invitro. As used herein, the phrase, “antisense RNA” refers to an RNAmolecule that is complementary to all or part of a target primarytranscript or mRNA and that blocks the expression of a target gene (U.S.Pat. No. 5,107,065). The complementarity of an antisense RNA may be withany part of the specific gene transcript, i.e., at the 5′ non-codingsequence, 3′ non-coding sequence, introns, or the coding sequence. Asused herein, the phrase, “functional RNA” refers to antisense RNA,ribozyme RNA, or other RNA that may not be translated but yet has aneffect on cellular processes. The terms “complement” and “reversecomplement” are used interchangeably herein with respect to mRNAtranscripts, and are meant to define the antisense RNA of the message.

As used herein, the term “single nucleotide polymorphism” or “SNP”refers typically, to a specific pair of nucleotides observed at a singlepolymorphic site. In some cases, which are rare, three or fournucleotides may be found.

As used herein, the term “splice variant” refers to a particular form ofa messenger RNA, wherein two splice variants share either (a) atranscriptional start site or (b) an open reading frame, but differ inthe inclusion/exclusion of one or more exons.

As used herein, the term “subject” refers to an animal, preferably amammal, including a human or non-human. The animal can be a domesticatedor non-domesticated animal.

As used herein, the term “treating” refers to reversing, alleviating,inhibiting the progress of, or preventing at least one overt symptomaticmanifestation of the disorder or condition to which such term applies,or one or more symptoms of such disorder or condition. The term“treatment” as used herein, refers to the act of treating, as “treating”is defined herein. For the present invention, the term “treat” means toalleviate or eliminate one or more symptoms, behavior or eventsassociated with a depressive disorder.

DEP2-1 Transcripts and Proteins Encoded Thereby

In one embodiment, the present invention relates to the discovery of anovel transcript of DEP2, named DEP2-1. FIG. 2 illustrates the isolatednucleic acid sequence for DEP2-1 (SEQ ID NO:2), which is 1198 base pairsin length. Nucleotides 1 to 316 of SEQ ID NO:2 comprise an exon that wasnot previously known to be in any naturally occurring transcript ofDEP2. The remaining portion of SEQ ID NO:2 (from nucleotides 317 to1198) are shared with LHPP and naturally occurring splice variantsthereof, and correspond to nucleotides 882 to 1760 of SEQ ID NO:9.DEP2-1 was initially assembled using Genecarta software (Compugen, TelAviv, Israel) from publicly available expressed sequence tags (“ESTs”).Specifically, the proprietary algorithms identified that certain ESTs(GenBank accession numbers BI669229, BI489679, BI756098, Z44231, R15274,R11923, BX952014, BI754006, H51555, H51378 and BG397886) each comprisedof sequences from within both nucleotides 1-316 and nucleotides 317-1198of SEQ ID NO:2. These two sequence blocks are not contiguous in thehuman genome, implying that SEQ ID NO:2 is a spliced transcript. Toconfirm that DEP2-1 is a naturally occurring transcript, four IMAGEclones (h5173309, h5194531, h5197955 and h4565014, corresponding toBI489679, BI756098, BI754006 and BG397886, respectively) were completelysequenced in both directions (see Example 5). The contiguous sequence ofnucleotides 19-1198 of SEQ ID NO:2 was thereby confirmed. IMAGE clonesh5173309, h5194531 and h5197955 are from brain, and include onlysequences shown in SEQ ID NO:2. Among the ESTs that assembled to formDEP2-1, all were from brain except for BG397886, which was from atonsillar primary B cell line. IMAGE clone h4565014 corresponds toBG397886. The sequence of this clone included nucleotides 299-1198followed by a polyadenine tail and a further 77 nucleotide sequence thatdid not match DEP2. Further, a single nucleotide polymorphism (1142G>A)was discovered at nucleotide 1142 of SEQ ID NO:2. Rapid amplification ofcDNA ends was performed to determine the 5′ end(s) of DEP2-1 (seeExample 6). These experiments identified two 5′ ends in human spinalcord RNA, at nucleotides 1 and 75 of SEQ ID NO:2. Two series of PCRexperiments were also performed to determine whether the first exon(nucleotides 1-315) of DEP2-1 is included in additional transcripts withupstream exons (see Example 7). The first experiments used forwardprimers in an upstream exon of LHPP and reverse primers in the firstexon of DEP2-1. The second experiments used forward primers in anupstream exon of LHPP and reverse primers in a downstream exon of LHPP.No sequence from the first exon of DEP2-1 was amplified in either set ofexperiments. In total, these experimental results demonstrate thatDEP2-1 is a naturally occurring transcript, that it is not analternative splice variant of LHPP, and thus does not encode a naturallyoccurring protein variant of Lhpp.

The isolated nucleic acid sequence of DEP2-1 has two coding regions,which are each illustrated in capital letters in FIG. 2. The firstcoding region (nucleotides 352 to 771 in SEQ ID NO:2) may encode for theprotein, referred to herein as Dep2-1a, shown in FIG. 3A (SEQ ID NO:3).Dep2-1a is 140 amino acids in length. The second coding region(nucleotides 812 to 1162 in SEQ ID NO:2) may encode for the protein,referred to herein as, Dep2-1b, shown in FIG. 3B (SEQ ID NO:4). Dep2-1bis 117 amino acids in length.

In addition, naturally occurring splice variants of DEP2-1 have beenidentified by the inventors of the present invention. These transcriptswere assembled using Genecarta software (Compugen, Tel Aviv, Israel)from publicly available expressed sequence tags (“ESTs”). These splicevariants of the DEP2-1 are shown in SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7 and SEQ ID NO:8. SEQ ID NO:5 is also shown in FIG. 4 and is 1092base pairs in length. The coding regions are shown in capital letters inthis figure. The first and second coding regions encode for Dep2-1a(See, FIG. 3A (SEQ ID NO:3)) and Dep2-1b (FIG. 3B (See, SEQ ID NO:4)).SEQ ID NO:5 was assembled on the basis of three ESTs (BG759116, BF976531and BQ706325), all of B cell origin. SEQ ID NO:6 is shown in FIG. 5 andis 1022 base pairs in length. The coding regions are shown in capitalletters in this figure. The first and second coding regions encode forDep2-1a (See, FIG. 3A (SEQ ID NO:3)) and Dep2-1b (FIG. 3B (See, SEQ IDNO:4)). SEQ ID NO:6 was assembled on the basis of two ESTs (BI226948 andBE396637), both from a Burkitt's lymphoma cell line. SEQ ID NO:7 isshown in FIG. 6 and is 1186 base pairs in length. The coding region isshown in capital letters in this figure. The coding region encodes forDep2-1b (See, FIG. 3A (SEQ ID NO:4)). SEQ ID NO:7 was assembled on thebasis of a single EST (CF454636), from the peripheral nervous system.SEQ ID NO:8 is shown in FIG. 7 and is 1005 base pairs in length. Thecoding region is shown in capital letters in this figure. The codingregion encodes for Dep2-1b (See, FIG. 3B (SEQ ID NO:4)). SEQ ID NO:8 wasassembled on the basis of a single EST (BE560698), from a Burkitt'slymphoma cell line.

The ESTs described in the preceding paragraph were used to assemble the5′ ends of the variant DEP2-1 transcripts. None of these ESTs containthe entire transcript sequence. In all cases, the 3′ end of each ofthese transcripts is common to all these sequences as well as to LHPP aswell as to some of the splice variants thereof and can be found inmultiple ESTs. These ESTs are listed below in Table B.

TABLE B GenBank Accession Tissue Source (If Specified) AA292585 Ovariantumor AA308083 Colon L KM12C (HCC) metastasis into mouse (liver)AA379353 Skin AA379626 Skin AA635531 Normal prostate AA669836 BoneMarrow stroma AA677990 Liver and spleen AA725685 AA912377 Dorsal rootganglion AI086359 Pooled human melanocyte, fetal heart, and pregnantuterus AI139589 Placenta AI97875 Anaplastic oligodendroglioma AI221142Pooled AI272203 Oligodendroglioma AI361781 B-cell, chronic lymphoticleukemia AI378120 B-cell, chronic lymphotic leukemia AI420571 ProstateAI439876 Lymphoma, follicular mixed small and large cell AI475774B-cell, chronic lymphotic leukemia AI492345 Kidney AI565021Well-defferentiated endometrial adenocarcinoma, 7 pooled tumors AI582637Kidney AI598057 Adenocarcinoma AI805094 Prostate AI972592 ProstateAW139347 AW301045 Moderately differentiated adenocarcinoma AW511836Moderately-differentiated endometrial adenocarcinoma, 3 pooled tumorsAW512618 Lymphoma, follicular mixed small and large cell AW954516BE675320 B-cell, chronic lymphotic leukemia BF740030 Kidney BF949274Nervous_normal BF986056 Placenta_normal BF986061 Placenta_normalBG027502 Osteosarcoma, cell line BG150734 Normal epithelium BI819728Pooled brain, lung, testis BQ644034 Hepatocellular carcinoma, cell lineBU539737 Adenocarcinoma, cell line BU683755 Primary lung cystic fibrosisepithelial cells BU753421 Placenta BX096697 Well-differentiatedendometrial adenocarcinoma, 7 pooled tumors BX330376 Placenta BX361464*Placenta BX423161 Adult brain CD631211 CD631212 CK823134*{circumflexover ( )} Islets of Langerhans CN265311 Embryonic stem cells, embryoidbodies derived from H1, H7 and H9 cells N38886 Multiple sclerosislesions *Reverse direction EST sequence also present in the publicdomain {circumflex over ( )}Two additional sequences from the same cloneare also present in the public domain.

It should be noted that the present invention also encompasses isolatednucleotide sequences (and the corresponding encoded proteins) havingsequences comprising, corresponding to, identical to, or complementaryto at least about 90% identity to SEQ ID NO:2. (All integers (andportions thereof) between 90% and 100% are also considered to be withinthe scope of the present invention with respect to percent identity.)For example, the present invention encompasses an isolated nucleic acidor fragment thereof comprising (a) a nucleotide sequence having at least90% identity to SEQ ID NO:2; or (b) a complement comprising a nucleotidesequence having at least 90% identity to SEQ ID NO:2. Such sequences maybe derived from any source, either isolated from a natural source, orproduced via a semi-synthetic route, or synthesized de novo.

The invention also includes a purified polypeptide that has at leastabout 90% amino acid similarity or identity to the amino acid sequencesof SEQ ID NO:3 or SEQ ID NO:4 of the above-noted proteins which are, inturn, encoded by the above-described nucleic acid sequences.

The present invention also encompasses an isolated nucleic acid sequencewhich encodes a polypeptide having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

Once DEP2-1 or any naturally occurring variants thereof have beenisolated, they may then be introduced into either a prokaryotic oreukaryotic host cell through the use of a vector or construct. Thevector, for example, a bacteriophage, cosmid or plasmid, may comprise anucleic acid sequence having a nucleotide sequence of SEQ ID NO:2, ornucleotides 352-771 or 812-1162 thereof, as well as any regulatorysequence (such as, but not limited to a promoter) which is functional inthe host cell and is able to elicit expression of the protein encoded bythe nucleotide sequence.

Alternatively, the vector may comprise a complement comprising anucleotide sequence of SEQ ID NO:2 or nucleotides 352-771 or 812-1162thereof, as well as any regulatory sequence. The regulatory sequence(for example, a promoter) is in operable association with, or operablylinked to, the sequence of SEQ ID NO:2, or nucleotides 352-771 or812-1162 thereof. Examples of promoters that can be used include LTR orthe SV40 promoter, the E. coli lac or trp, the phage lambda P sub Lpromoter and other promoters known to those of skill in the art.Additionally, nucleic acid sequences which encode other proteins,oligosaccharides, lipids, etc. may also be included within the vector aswell as other regulatory sequences such as a polyadenylation signal (forexample, the poly-A signal of SV-40T-antigen, ovalalbumin or bovinegrowth hormone). The choice of sequences present in the construct isdependent upon the desired expression products as well as the nature ofthe host cell.

Once the vector has been constructed, it can be introduced (namely,transformed or transfected) into host cells, such as mammalian (such as,but not limited to, simian, canine, feline, bovine, equine, rodent,murine, etc.) or non-mammalian (such as, but not limited to, insect,reptile, fish, avian, etc.) cells, using any method known to those ofskill in the art including, but not limited to, electroporation, calciumphosphate precipitation, DEAE dextran, lipofection, and receptormediated endocytosis, polybrene, particle bombardment, andmicroinjection. Alternatively, the vector can be delivered to the cellas a viral particle (either replication competent or deficient).Examples of viruses useful for the delivery of nucleic acid include, butare not limited to, lentivirus, adenoviruses, adeno-associated viruses,retroviruses, herpesviruses, and vaccinia viruses. Other virusessuitable for delivery of nucleic acid sequences into cells that areknown to those of skill in the art may be equivalently used in thepresent invention.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating the promoter sequences, selectingtransfected cells, etc. The culture conditions, such as temperature, pHand the like, are those previously used with the host cell selected forexpression, and will be apparent to those of skill in the art.

The engineered host cells containing the incorporated vector(s) can beidentified using hybridization techniques that are well known to thoseof skill in the art or by using the polymerase chain reaction to amplifyspecific polynucleotide sequences. If the nucleic acid sequencetransferred to the cells produces a protein that can be detected, forexample, by means of an immunological or enzymatic assay, then thepresence of recombinant protein can be confirmed by performing theassays either on the medium surrounding the cells or on cellularlysates.

Non-Human Transgenic Animals

In another embodiment, the present invention relates to non-humantransgenic animals that contain the transcripts that arise from DEP2 aswell as methods of making said animals. Specifically, the nucleic acidsequences that can be used in said non-human transgenic animals include:(a) LHPP (SEQ ID NO:9); (b) naturally occurring splice variants of LHPP(SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:26; (c) DEP2-1(SEQ ID NO:2); (d) naturally occurring splice variants of DEP2-1 (SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8); (e) DEP2-2 (SEQ IDNO:28); (f) DEP2-3 (SEQ ID NO:30); (g) GenBank sequence AK127935 (SEQ IDNO:31); and (h) GenBank sequence AW867792 (SEQ ID NO:33).

A variety of methods can be used to create the non-human transgenicanimals. For example, the generation of a specific alteration of anucleic acid sequence of a target gene is one approach that can be used.Alterations can be accomplished by a variety of enzymatic and chemicalmethods used in vitro. One of the most common methods uses a specificoligonucleotide as a mutagen to generate precisely designed deletions,insertions and point mutations in a target gene. Secondly, a wildtypehuman gene or humanized non-human animal gene could be inserted byhomologous recombination. It is also possible to insert an altered ormutated (singly or multiply) human gene as genomic or minigeneconstructs.

Additionally, non-human transgenic animals can also be made wherein atleast one endogenous target gene is “knocked-out”. The creation ofknock-out animals allows those of skill in the art to assess in vivofunction of the gene that has been “knocked-out”. The knock-out of atleast one target gene may be accomplished in a variety of ways. Onestrategy that can be used to “knock-out” a target gene is by theinsertion of artificially modified fragments of the endogenous gene byhomologous recombination. In this technique, mutant alleles areintroduced by homologous recombination into embryonic stem (“ES”) cells.The embryonic stem cells containing a knock out mutation in one alleleof the gene being studied are introduced into a blastocyst. Theresultant animals are chimeras containing tissues derived from both thetransplanted ES cells and host cells. The chimeric animals are mated toassess whether the mutation is incorporated into the germ line. Thosechimeric animals each heterozygous for the knock-out mutation are matedto produce homozygous knock-out mice. A second strategy that can be usedto “knock-out” at least one gene involves using siRNA and shRNA andoocyte microinjection or transfection or microinjection into embryonicstem cells as described further herein.

The present invention contemplates that the somatic and germ cells ofsaid non-human transgenic animal comprise an exogenous and stablytransmitted nucleic acid sequence of SEQ ID NO:2 (DEP2-1). Additionally,the present invention further contemplates that the somatic and germcells of the transgenic animals comprise an exogenous and stablytransmitted nucleic acid sequence having a nucleotide sequence selectedfrom the group consisting of: SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO: 22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 andSEQ ID NO:33, with the proviso that its somatic and germ cells do notcomprise an exogenous and stably transmitted nucleic acid having anucleotide sequence of SEQ ID NO:2. The methods for creating suchtransgenic animals will be discussed in more detail below.

The present invention further contemplates non-human transgenic animalswherein a nucleic acid comprising a nucleotide sequence of SEQ ID NO:2(DEP2-1) is knocked out in said animal. Additionally, the presentinvention contemplates a non-human transgenic animal wherein a nucleicacid having a nucleotide sequence selected from the group consisting of:SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:26, SEQID NO:28, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:33 is knocked out,with the proviso that a nucleic acid sequence of SEQ ID NO:2 is notmodified or altered. The methods for creating such “knock-out” animalswill be described in more detail below.

To create a non-human transgenic animal containing an exogenous andstably transmitted nucleic acid of a target gene or other nucleic acidsequence, a nucleic sequence of interest can be inserted into anon-human animal germ line using standard techniques of oocytemicroinjection or transfection or microinjection into embryonic stemcells. Alternatively, if it is desired to knock-out or replace anendogenous gene, homologous recombination using embryonic stem cells orsiRNA or shRNA using oocyte microinjection or transfection ormicroinjection of embryonic stem cells can be used.

For oocyte injection, at least one nucleic acid sequence of interestthat is operably linked to the promoter can be inserted into thepronucleus of a just-fertilized non-human animal oocyte. This oocyte isthen reimplanted into a pseudopregnant foster mother. The livebornnon-human animal can then be screened for integrants by analyzing theanimal's DNA (using polymerase chain reaction for example) such as fromthe tail, for the presence of the polynucleotide sequence of interest.Chimeric non-human animals are then identified. The nucleic acid can bea complete genomic sequence injected as a YAC or chromosome fragment, acDNA, or a minigene containing the entire coding region and otherelements found to be necessary for optimum expression.

Retroviral or lentiviral infection (See, Lois C, et al., Science,295:868-872 (2002) (which teaches methods for transgenics usinglentiviral transgenesis)) of early embryos can also be done to insert analtered gene. In this method, the altered gene is inserted into aretroviral vector which is used to directly infect mouse embryos duringthe early stages of development to generate a chimera, some of whichwill lead to germline transmission (Jaenisch, R., Proc. Natl. Acad. Sci.USA, 73: 1260-1264 (1976)).

Homologous recombination using embryonic stem cells allows for thescreening of gene transfer cells to identify the rare homologousrecombination events. Once identified, these can be used to generatechimeras by injection of at least one non-human animal blastocyst and aproportion of the resulting animals will show germline transmission fromthe recombinant line. This gene targeting methodology is especiallyuseful if inactivation of the gene is desired. For example, inactivationof the gene can be done by designing a polynucleotide fragment whichcontains sequences from an exon flanking a selectable marker. Homologousrecombination leads to the insertion of the marker sequences in themiddle of an exon, inactivating the gene. DNA analysis of individualclones can then be used to recognize the homologous recombinationevents.

Alternatively, “knock-out” of a target gene can be accomplished usingsiRNA or shRNA. In one strategy, oocyte microinjection can be used asdescribed herein. Specifically, at least one nucleic acid sequence ofinterest that expresses at least one RNA molecule that is siRNA orshRNA, and that is operably linked to at least one promoter (such as aRNA pol III dependent promoter), is prepared using the methods describedherein. This nucleic acid is introduced into a non-human animalfertilized oocyte, preferably by injection. The fertilized oocyte isthen allowed to develop into an embryo. The resulting embryo is thentransferred into a pseudopregnant female non-human animal and thenallowed to give birth. Liveborn non-human animals are then screened forchimeric animals that contain the nucleic acid by obtaining a sample andanalyzing the animal's DNA (using techniques such as polymerase chainreaction) and such chimeric non-human animals are identified. When thesenon-human animals are treated with an inducing agent, transcription isinduced, the siRNA or shRNA expressed, and the target gene is repressedor “knocked-out”. In the absence of the inducing agent, the gene is notrepressed or “knocked-out”.

In a second strategy, microinjection of embryonic stem cells can be usedas described herein. Specifically, at least one nucleic acid sequence ofinterest that expresses at least one RNA molecule that is siRNA orshRNA, and is operably linked to at least one RNA pol III dependentpromoter sequence of the present invention, is prepared using themethods described herein. This nucleic acid is introduced into non-humananimal embryonic stem cells which can be used to generate chimeras byintroducing these embryonic stem cells, preferably by injection, into atleast one non-human animal blastocyst. The resulting blastocyst is thenimplanted into a pseudopregnant female non-human animal and then allowedto give birth to a chimeric non-human animal PCR can be used to identifythe animals of interest. Liveborn non-human animals are then screenedfor chimeric animals that contain the nucleic acid by obtaining andanalyzing a sample of said animal's DNA (using techniques such aspolymerase chain reaction) and such chimeric non-human animals areidentified. This chimeric non-human animal can then be used in breedingto produce a transgenic non-human animal that stably contain thisnucleic acid within their genome. As with the previous method, whenthese non-human animals are treated with an inducing agent,transcription is induced, the siRNA or shRNA expressed, and the targetgene is repressed or “knocked-out”. In the absence of the inducingagent, the gene is not repressed or “knocked-out”.

Methods of making transgenic animals are described, for example, in Wallet al., J. Cell Biochem., 49(2):113-20 (1992); Hogan, et al.,“Manipulating the mouse embryo”, A Laboratory Manual. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1992); in WO 91/08216 orU.S. Pat. No. 4,736,866 the disclosures of which are hereby incorporatedby reference in their entirety.

Method of Modifying or Altering Expression of Nucleic Acid Molecules

In another embodiment, the present invention relates to methods ofmodifying or altering the expression of nucleic acid sequences. Thepresent invention contemplates that the nucleic acid sequence whoseexpression is modified or altered is SEQ ID NO:2. The present inventionfurther contemplates that the nucleic acid sequence whose expression ismodified or altered is a nucleic acid having a nucleotide sequence of atleast one of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO:24, SEQID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:33.

Methods for modifying or altering the expression of a nucleic acidsequence are well known to those skilled in the art. Specifically, saidmethods involve exposing a cell or administering to a subject (such as atransgenic non-human animal (for example, a transgenic non-human animalhaving at least one nucleic acid molecule knocked-out)) containing anucleic acid whose expression is to be modified or altered at least onenucleic acid molecule. The methods described herein could be useful,such as in transgenic non-human animals (such as in transgenic non-humananimals having at least one nucleic acid molecule knocked-out), asanimal models for major depression or a related disorder. Nucleic acidmolecules such as antisense molecules, aptamers, triplexing agents,ribozymes, siRNA, or co-suppression (co-suppressor) RNA can be used insaid methods.

An antisense molecule, aptamer, triplexing agent, ribozyme or siRNA areDNA, RNA or chemically modified or hybrid sequences thereof of varyinglength that are single or double stranded. These nucleic acid moleculesare complementary to a target nucleic acid sequence, such as a mRNA of anucleic acid (a) having a nucleotide sequence of SEQ ID NO:2; or (b) ofat least one of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14,SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO:24,SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:33,and can be a coding sequence, a polynucleotide sequence comprising anintron-exon junction, a regulatory sequence, such as a promotersequence, or the like. The degree of complementarity is such that thenucleic acid molecule can interact specifically with the target nucleicacid sequence in a cell. Depending on the total length of the nucleicacid molecule, one or a few mismatches with respect to the targetnucleic acid sequence can be tolerated without losing the specificity ofthe nucleic acid molecule for the target sequence. Thus, a fewmismatches, if any, would be tolerated, for example, in an antisensemolecule containing, for example, 20 consecutive nucleotides, whereasseveral mismatches will not affect the hybridization efficiency of anantisense molecule that is complementary to a full length of a targetmRNA encoding a protein (such as, Dep2-1a, Dep2-1b, Dep2-2, Dep2-4 orDep2-5). The number of mismatches that can be tolerated can beestimated, using well known formulas for determining hybridizationkinetics (See, Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) Edition (1989)) or can be determined empirically using methodsknown in the art, particularly by determining that the presence of theantisense molecule, aptamer, triplexing agent, ribozyme or siRNA in acell modifies or alters (such as by decreasing) the level of expressionof the target sequence in a cell.

A nucleic acid molecule useful as an antisense molecule, aptamer,triplexing agent, ribozyme or siRNA can reduce or inhibit translation orcleave a target nucleic acid, thereby reducing or inhibiting the amountof the protein encoded by said target nucleic acid in a cell. Forexample, an antisense molecule can bind to an mRNA to form a doublestranded molecule that cannot be translated in a cell. Antisenseoligonucleotides of about 15 to 50 consecutive nucleotides are preferredsince they are easily synthesized and can hybridize specifically with atarget nucleic acid, although longer antisense molecules can be used.When the antisense molecule is contacted directly with a target cell, itcan be operatively associated with a chemically reactive group such as,but not limited to, iron-linked EDTA, which cleaves a target RNA at thesite of hybridization. A triplexing agent, in comparison, can stalltranscription (Maher et al., Antisense Res. Devel., 1:227 (1991);Helene, Anticancer Drug Design, 6:569 (1991)). Aptamers adopt highlyspecific three-dimensional conformations that enable them to bind to aspecific location on a molecule whose activity is being affected.Methods for making antisense molecules, aptamers and triplexing agentsare well known in the art.

A ribozyme is a catalytic RNA molecule that cleaves RNA in asequence-specific manner. Ribozymes that cleave themselves are calledcis-acting ribozymes, while ribozymes that cleave other RNA moleculesare called trans-acting ribozymes. Nucleic acids molecules can encoderibozymes designed to cleave particular mRNA transcripts, thuspreventing expression of a polypeptide. A ribozyme sequence can have asequence from a hammerhead, axhead, or hairpin ribozyme, and may bemodified to have either slow cleavage activity or enhanced cleavageactivity. For example, nucleotide substitutions can be made to modifycleavage activity (see, e.g., Doudna and Cech, Nature, 418:222-228(2002)). Hammerhead ribozymes are useful for destroying particularmRNAs, although various ribozymes that cleave mRNA at site-specificrecognition sequences can be used. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target RNAcontain a 5′-UG-3′ nucleotide sequence. The construction and productionof hammerhead ribozymes is known in the art. See, for example, U.S. Pat.No. 5,254,678. Hammerhead ribozyme sequences can be embedded in a stableRNA such as a transfer RNA (tRNA) to increase cleavage efficiency invivo (Perriman, R. et al., Proc. Natl. Acad. Sci. USA, 92(13):6175-6179(1995); de Feyter, R. and Gaudron, J., Methods in Molecular Biology,Vol. 74, Chapter 43, “Expressing Ribozymes in Plants”, Edited by Turner,P. C, Humana Press Inc., Totowa, N.J.).

siRNA useful in the present invention can be obtained, for example,using an in vitro transcription system or can be synthesized chemically,and can be contacted with cells (or administered to a subject) as RNAmolecules. siRNA also can be expressed from an encoding nucleic acid,which can be contacted with cells (or administered to a subject). siRNAscan be designed using techniques well known to those skilled in the art.

Another nucleic acid molecule that is useful in the present methods alsocan be a co-suppression RNA that reduces or inhibits transcription of atarget nucleic acid, such as a nucleic acid (a) having a nucleotidesequence of SEQ ID NO:2; or (b) of at least one of SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:31 or SEQ ID NO:33. A co-suppressor RNA, like an siRNA,comprises (or encodes) an RNA comprising an inverted repeat, whichincludes a first oligonucleotide that selectively hybridizes to thetarget nucleic acid or gene and, in operative linkage, a secondoligonucleotide that is complementary and in a reverse orientation tothe first oligonucleotide. In comparison to an siRNA, which comprises afunctional portion of a transcribed region of the target nucleic acid ortarget gene and reduces or inhibits translation of RNA transcribed fromthe nucleic acid or gene, a co-suppressor RNA comprises a functionalportion of a transcriptional regulatory region of the target nucleicacid or gene (namely, a promoter region) and reduces or inhibitstranscription of the nucleic acid or gene. Methods for makingco-suppression RNA are well known in the art.

Polymorphism Detection/Genotyping

In another embodiment, the present invention relates methods ofgenotyping one or more subjects. The information obtained from thegenotyping of subjects can be used in a variety of different ways. Forexample, the genotyping of subjects can be used to diagnose thosesubjects suffering from major depression or a related disorder or atrisk of developing major depression or a related disorder, provide aprognosis for or predict or diagnose a response to treatment for asubject suffering from major depression or a related disorder, oridentify subjects for selection or inclusion in a clinical trial fortreating major depression or a related disorder. Additionally, genotypescan be used to analyze the results of a clinical trial for subjectsbeing treated for major depression or a related disorder. Specifically,the relationship the genotypes of subjects and the clinical outcome ofsaid subjects can be determined.

Genotyping involves obtaining a test sample from said subject(s). Thesubject may or may not be experiencing any symptoms of major depressionor a related disorder at the time the test sample is obtained. In thisembodiment, a test sample is any biological sample which contains theDNA of the subject. Test samples can be prepared using techniques wellknown to those skilled in the art such as by obtaining a specimen froman individual and, if necessary, disrupting any cells contained thereinto release DNA. Examples of test samples include, but are not limitedto, whole blood, serum, plasma, cerebrospinal fluid, sputum, bronchialwashing, bronchial aspires, urine, lymph fluids, and various externalsecretions of the respiratory, intestinal and genitourinary tracts,tears, saliva, milk, white blood cells, myelomas and the like, etc.

Once the test sample(s) is obtained, it is analyzed, using routinetechniques known in the art, in order to determine the presence orabsence of specific sequences (alleles) for: (a) at least onepolymorphic site in nucleotides 1 to 316 of SEQ ID NO:2; (b) a T-Cpolymorphism at position (nucleotide) 136 of SEQ ID NO:2; (c) a A-Gpolymorphism at position 210 of SEQ ID NO:2; (d) a G-A polymorphism atposition 242 of SEQ ID NO:2; (e) at least one polymorphic site selectedfrom nucleotides 77402 and 79906 of SEQ ID NO:1; (f) at least onepolymorphic site in SEQ ID NO:1; or (g) any combinations of (a)-(f).Additionally, the test sample may optionally be further analyzed for aC-G polymorphism at position −1019 of a human serotonin receptor 1A gene(“HTR1A”). For example, the identification of at least one polymorphicsite at nucleotide 38048, 77402 or 79906 in SEQ ID NO:1 in combinationwith the identification of a C-G polymorphism at position −1019 of ahuman serotonin receptor 1A gene in a test sample obtained from asubject may indicate that the subject is at risk of developing majordepression or a related disorder.

The genotype of the subject can be determined based on the combinationof sequences present at one of more polymorphic sites. Once the genotypeof the subject has been determined, then further determinations can bemade, such as, diagnosing whether the subject has major depression or arelated disorder or is at risk of developing major depression or arelated disorder, providing a prognosis for or predicting the responseto treatment for a subject having major depression or a relateddisorder, determining whether the subject should be selected forinclusion in a clinical trial for treatment of major depression or arelated disorder, or analyzing the relationship between genotypes ofsubjects and their clinical outcome. Additionally, if the test sample isalso analyzed for the presence of sequences at a C-G polymorphism atposition −1019 in the HTR1A gene, then the genotype(s) at one or morepolymorphic sites in DEP2 may be used in combination with the genotypeat HTR1A to make further determinations, as elaborated above.

As briefly discussed above, techniques for identifying the presence orabsence of at least one sequence (allele) at a polymorphic site in atest sample are well known in the art and include, but are not limitedto direct sequencing, amplification, fragment length polymorphismassays, mobility based assays, hybridization assays and massspectroscopy. These techniques will be discussed briefly below.

Direct Sequencing

The presence or absence of a sequence at a polymorphic site may bedetermined by direct nucleotide sequencing. Methods for directsequencing are known in the art. For example, following amplification ofthe DNA from the test sample, the DNA can be sequenced using manualsequencing techniques, such as those that employ radioactive markernucleotides, or by automated sequencing. The results of the sequencingcan be displayed using any suitable method known in the art. Thesequence is examined and the presence or absence of a given sequence ata polymorphic site is determined.

Amplification

The presence or absence of a sequence at a polymorphic site may bedetermined using amplification techniques, such as PCR. PCR involves theuse of primers to amplify a region of a DNA sequence from the testsample containing the polymorphic site of interest. The design ofprimers is well known to those skilled in the art. For example, primerscan be designed that hybridize only to a portion of SEQ ID NO:1 or aportion of SEQ ID NO:2 (hereinafter “the wildtype”). If these wildtypeprimers result in a PCR product, then the subject has the wildtypeallele (namely, SEQ ID NO:1 or SEQ ID NO:2). Similarly, primers can bedesigned that hybridize only to a portion of SEQ ID NO:1 or a portion ofSEQ ID NO:2 containing a variant sequence at one or more polymorphicsites (hereinafter “the variant”). If these variant primers result in aPCR product, then the subject has the variant allele (namely, SEQ IDNO:1 or SEQ ID NO:2). The presence of an amplification product only whenwildtype primers are used, or only when variant primers are used,indicate a homozygous wildtype or variant genotype, respectively. Thepresence of an amplification product when either wildtype or variantprimers are used indicates a heterozygous genotype. Amplificationmethods other than PCR can be used. Such methods include stranddisplacement, the QB replicase system, the repair chain reaction, ligasechain reaction, rolling circle amplification and ligation activatedtranscription.

Fragment Length Polymorphism Assays

The presence or absence of sequences at a polymorphic site may bedetermined using a fragment length polymorphism assay. In a fragmentlength polymorphism assay, a unique DNA banding pattern based oncleaving the DNA at a series of positions is generated using an enzyme(such as, but not limited to, a restriction endonuclease). DNA fragmentsfrom the test sample containing a variant sequence will have a differentbanding pattern than DNA fragments generated from the wildtype.

For example, sequences at a polymorphic site can be detected using arestriction fragment length polymorphism assay (“RFLP”). The region ofinterest in the DNA is first isolated using PCR. The PCR products arethen cleaved with restriction enzymes known to give a unique lengthfragment for a given variant sequence. The restriction-enzyme digestedPCR products are separated and detected (such by gel electrophoresis)and visualized (such as, but not limited to, by ethidium bromidestaining). The length of the fragments is compared to molecular weightmarkers or fragments generated from wildtype and variant controls (forexample, vectors containing the wildtype and variant sequences,respectively).

Sequences (alleles) at a polymorphic site can also be detected using aCLEAVASE fragment length polymorphism assay (“CFLP”; Third WaveTechnologies, Madison, Wis.; See, U.S. Pat. No. 5,888,780). This assayis based on the observation that when single strands of DNA fold onthemselves, they assume higher order structures that are highlyindividual to the precise sequence of the DNA molecule. These secondarystructures involve partially duplexed regions of DNA such that singlestranded regions are juxtaposed with double stranded DNA hairpins. TheCLEAVASE I enzyme, is a structure-specific, thermostable nuclease thatrecognizes and cleaves the junctions between these single-stranded anddouble-stranded regions.

The region of interest is first isolated using routine techniques knownin the art, such as by PCR. Next, DNA strands are separated by heating.The reactions are cooled to allow intrastrand secondary structure toform. The PCR products are then treated with the CLEAVASE I enzyme togenerate a series of fragments that are unique to a given wildtype orvariant sequence. The CLEAVASE enzyme treated PCR products are separatedand detected (such by gel electrophoresis) and visualized (such as, butnot limited to, by ethidium bromide staining). The length of thefragments is compared to molecular weight markers or fragments generatedfrom wild-type and variant controls.

Mobility Based Assays

The presence or absence of a sequence (allele) at a polymorphic site maybe determined by a single strand conformation polymorphism assay(“SSCP”). In this technique, PCR products from the region to be testedare heat denatured and rapidly cooled to avoid the reassociation ofcomplementary strands. The single strands then form sequence dependentconformations that influence electrophoretic mobility. The differentmobilities can then be analyzed by electrophoresis.

Alternatively, the assessment of a polymorphism may be by a heteroduplexassay. In this analysis, the DNA sequence to be tested is amplified,denatured and renatured to itself or to known wildtype DNA (namely, fromSEQ ID NO:1 or SEQ ID NO:2). Heteroduplexes between different allelescontain DNA “bubbles” at mismatched basepairs that can affectelectrophoretic mobility. Therefore, electrophoresis can be used toindicate the presence or absence of wildtype and variant sequences.

Hybridization Assays

The presence or absence of a sequence (allele) at a polymorphic site canbe detected in a hybridization assay. In a hybridization assay, thepresence or absence of a given sequence (allele) is determined based onthe ability of the DNA from the test sample to hybridize to acomplementary DNA molecule (such as, but not limited to, a probe). Thehybridization of a probe to DNA from the test sample is subsequentlydetected. Detection of hybridization only to a wildtype probe, or onlyto a variant probe, indicate a homozygous wildtype or variant genotype,respectively. Detection of hybridization to both wildtype and variantprobes indicates a heterozygous genotype. A number of hybridizationassays using a variety of technologies for hybridization and detectionare available. Examples of some of these assays are provided below.

Solution Based Detection

The presence or absence of polymorphisms can be determined using anysolution based detection techniques known in the art. An example of sucha technique that can be used is TaqMan® (Applied Biosystems, ForestCity, Calif.; see, Holland et al; Proc. Natl. Acad. Sci. USA88:7276-7280 (1991); and Gelmini et al. Clin. Chem. 43:752-758 (1997)).TaqMan® allows for the real-time quantification of PCR. TaqMan® probesare widely commercially available, and the TaqMan® system (AppliedBiosystems) is well known in the art. TaqMan® probes anneal between theupstream and downstream primer in a PCR reaction. They contain a5′-fluorophore and a 3′-quencher. During amplification the 5′-3′exonuclease activity of the Taq polymerase cleaves the fluorophore offthe probe. Since the fluorophore is no longer in close proximity to thequencher, the fluorophore will be allowed to fluoresce. The resultingfluorescence may be measured, and is in direct proportion to the amountof target sequence that is being amplified.

Another technique that can be used is a Molecular Beacon (See, Tyagi etal., Nat. Biotechnol. 14:303-308 (1996); and Tyagi et al., Nat.Biotechnol. 16:49-53 (1998)), the beacons are hairpin-shaped probes withan internally quenched fluorophore whose fluorescence is restored whenbound to its target. The loop portion acts as the probe while the stemis formed by complimentary “arm” sequences at the ends of the beacon. Afluorophore and quenching moiety are attached at opposite ends, the stemkeeping each of the moieties in close proximity, causing the fluorophoreto be quenched by energy transfer. When the beacon detects its target,it undergoes a conformational change forcing the stem apart, thusseparating the fluorophore and quencher. This causes the energy transferto be disrupted to restore fluorescence. Any suitable fluorophore knownin the art can be used. For example, fluorophores that can be usedinclude, but are not limited to, FAM, HEX®, NED®, ROX®, Texas Red®.Quenchers that can be used include, but are not limited to, Dabcyl andTAMRA.

Another technique that can be used is Pyrosequencing™ (Pyrosequencing,Inc. Westborough, Mass.). This technique is based on the hybridizationof a primer to a single stranded, PCR-amplified, DNA template in thepresence of DNA polymerase, ATP sulfurylase, luciferase and apyraseenzymes and the adenosine 5′ phosphosulfate (“APS”) and luciferinsubstrates. In the second step, the first of four deoxynucleotidetriphosphates (“DCNTP”) is added to the reaction and the DNA polymerasecatalyzes the incorporation of the deoxynucleotide triphosphate into theDNA strand, if it is complementary to the base in the template strand.Each incorporation event is accompanied by release of pyrophosphate(“PPi”) in a quantity equimolar to the amount of incorporatednucleotide. In the last step, the ATP sulfurylase quantitativelyconverts PPi to ATP in the presence of adenosine 5′-phosphosulfate. ThisATP drives the luciferase-mediated conversion of luciferin tooxyluciferin that generates visible light in amounts that areproportional to the amount of ATP. The light produced in theluciferase-catalyzed reaction is detected by a charge coupled device(‘CCD”) camera and seen as a peak in a Pyrogram™. Each light signal isproportional to the number of nucleotides incorporated.

Detection of Hybridization Using Reverse Solid Phase Detection

The presence or absence of polymorphisms can also be determined usingreverse solid phase detection, such as, but not limited to, amicroarray, such as a DNA chip assay. In a DNA chip assay, a series ofprobes are affixed to a solid support. The probes are designed to beunique to a given polymorphism. The DNA obtained from the test sample iscontacted with the DNA “chip” and hybridization is detected. Any DNA“chip” assay known in the art can be used in the methods of the presentinvention. For example, the DNA chip assay can be a GeneChip assay(Affymetrix, Santa Clara, Calif.; See, U.S. Pat. No. 6,045,996). TheGeneChip technology uses miniaturized, high-density arrays of probesaffixed to a “chip.” Alternatively, a DNA microchip containingelectronically captured probes (Nanogen, San Diego, Calif.; See, U.S.Pat. No. 6,068,818) can be used. Also, a “bead array” can also be used(Illumina, San Diego, Calif.; See WO 99/67641 and WO 00/39587). Illuminauses a BEAD ARRAY technology that combines fiber optic bundles and beadsthat self-assemble into an array.

Solid Phase Detection

In solid phase detection, hybridization of a probe to the sequence ofinterest, such as a polymorphism, is detected directly by visualizing abound probe by using Southern blotting. In this technique, genomic DNAis isolated from a subject. The DNA is then cleaved with a series ofrestriction enzymes that cleave infrequently in the genome and not nearany of the markers being assayed. The DNA is then separated (such as,but not limited to, by agarose gel electrophoresis) and transferred to amembrane. At least one probe which has been labeled with, for example, aradioactive, fluorescent or enzymatic label, specific for thepolymorphism being detected is allowed to contact the membrane under acondition of low, medium, or high stringency conditions. Unbound probeis removed and the presence of binding is detected by visualizing thelabeled probe.

Enzymatic Detection of Hybridization

The presence or absence of polymorphisms can be detected using an assaythat detects hybridization by enzymatic cleavage of specific structures(“INVADER assay”, Third Wave Molecular Diagnostics, Madison, Wis.; See,U.S. Pat. No. 6,001,567). The INVADER assay detects specific DNA and RNAsequences by using structure-specific enzymes to cleave a complex formedby the hybridization of overlapping probes. Elevated temperature and anexcess of one of the probes enable multiple probes to be cleaved foreach target sequence present without temperature cycling. These cleavedprobes then direct cleavage of a second labeled probe. The secondaryprobe can be 5′-end labeled (such as, but not limited to, withfluorescein) that is quenched by an internal dye. Upon cleavage, thedequenched fluorescein labeled product may be detected using a standardfluorescence plate reader.

The INVADER assay detects specific mutations and SNPs in unamplifiedgenomic DNA. The isolated DNA sample is contacted with the first probespecific either for a SNP or wild type sequence and allowed tohybridize. Then a secondary probe, specific to the first probe, andcontaining the fluorescein label, is hybridized and the enzyme is added.Binding is detected using a fluorescent plate reader and comparing thesignal of the test sample to known positive and negative controls.

Hybridization of a bound probe can be detected using a TaqMan® assayusing the techniques described previously herein.

In still further embodiments, polymorphisms are detected using anysingle base extension (“SBE”) methods known in the art (See U.S. Pat.Nos. 5,888,819 and 6,004,744). For example, a shifted termination assay(“STA”) can be performed. The STA method involves designing a detectionprimer that is complementary to a target DNA. The detection primer islabeled with any detectable label known in the art. The 3′-terminal ofdetection primer ends at the base just before the target base. Thedetection primer hybridizes to the target nucleic acid sequence. Whenperforming a primer extension reaction, if the first base is the targetbase, a primer extension reaction will be terminated at the target baseposition without incorporating any of the labeled nucleotides. No colorreaction will be detected. If the target base is changed by any type ofmutation, including point mutation (SNP), deletion, insertion, andtranslocation, a primer extension reaction will continue through thetarget base position, and multiple labeled nucleotides will beincorporated into the extended detection primer. A strong color reactionwill be observed. A STA can be performed on a DNA sequence or usingfluorescence polarization.

Another SBE that can be performed is a SNP-IT primer extension assay(Orchid Biosciences, Princeton, N.J.; See, U.S. Pat. No. 5,952,174). Inthis assay, SNPs are identified using a specially synthesized DNA primerand a DNA polymerase to selectively extend the DNA chain by one base atthe suspected SNP location. DNA in the region of interest is amplifiedand denatured. PCR is then performed using miniaturized systems calledmicrofluidics. Detection is accomplished by adding a label to thenucleotide suspected of being at the polymorphic site. Incorporation ofthe label into the DNA can be detected by any method known in the art.

Mass Spectroscopy

The presence or absence of polymorphisms can be detected using aMassARRAY system (Sequenom, San Diego, Calif.; See, U.S. Pat. No.6,043,031). DNA is isolated from test samples using routine proceduresknown to those skilled in the art. Next, specific DNA regions containingthe polymorphism of interest, about 200 base pairs in length, areamplified by PCR. The amplified fragments are then attached by onestrand to a solid surface and the non-immobilized strands are removed bystandard denaturation and washing. The remaining immobilized singlestrand then serves as a template for automated enzymatic reactions thatproduce genotype specific diagnostic products.

Very small quantities of the enzymatic products, typically five to tennanoliters, are then transferred to a SpectroCHIP array for subsequentautomated analysis with the SpectroREADER mass spectrometer. Each spotis preloaded with light absorbing crystals that form a matrix with thedispensed diagnostic product. The MassARRAY system uses MALDI-TOF(Matrix Assisted Laser Desorption Ionization-Time of Flight) massspectrometry. In a process known as desorption, the matrix is hit with apulse from a laser beam. Energy from the laser beam is transferred tothe matrix and it is vaporized resulting in a small amount of thediagnostic product being expelled into a flight tube. As the diagnosticproduct is charged when an electrical field pulse is subsequentlyapplied to the tube they are launched down the flight tube towards adetector. The time between application of the electrical field pulse andcollision of the diagnostic product with the detector is referred to asthe time of flight. This is a very precise measure of the product'smolecular weight, as a molecule's mass correlates directly with time offlight with smaller molecules flying faster than larger molecules. Theentire assay is completed in less than 0.0001 second, enabling samplesto be analyzed in a total of 3-5 second including repetitive datacollection. The SpectroTYPER software then calculates, records, comparesand reports, the genotypes at the rate of three seconds per sample.

Kits

The present invention also provides kits that enable or allow for thedetection of a genotype of one or more subjects. These kits are usefulfor diagnosing those subjects suffering from major depression or arelated disorder or at risk of developing major depression or a relateddisorder, providing a prognosis for or predicting a response totreatment for a subject suffering from major depression or a relateddisorder, identifying subjects for selection or inclusion in a clinicaltrial for treating major depression or a related disorder, or foranalyzing the relationship between genotypes of subjects being treatedfor major depression or a related disorder and their clinical outcome.

The kits can be produced in a variety of ways. For example, the kitscontain at least one reagent useful for detecting (a) at least onepolymorphic site in SEQ ID NO:1; (b) at least one polymorphic site innucleotides 1 to 316 of SEQ ID NO:2; (c) a T-C polymorphism at position136 of SEQ ID NO:2; (d) a A-G polymorphism at position 210 of SEQ IDNO:2; (e) a G-A polymorphism at position 242 of SEQ ID NO:2; (f) atleast one polymorphic site in SEQ ID NO:1; (g) a polymorphic site innucleotide 77402 of SEQ ID NO:1; (h) a polymorphic site in nucleotide79906 in SEQ ID NO:1; or (i) any combinations of (a)-(h). Additionally,any of the kits described above in (a)-(i) can further contain at leastone reagent useful for detecting a C-G polymorphism at position −1019 ina human serotonin receptor 1A gene. Examples of the at least one reagentthat can be included in the kits described herein are one or moreprimers for amplifying the region of DNA containing the polymorphic siteor one or more probes that bind to or near the polymorphic site. Inaddition, the kits can further contain (a) instructions for determiningthe genotype of a subject; (b) ancillary reagents such as bufferingagents, nucleic acid stabilizing reagents, protein stabilizing reagents,and signal producing systems (such as, but not limited to, fluorescencegenerating systems); or (c) positive and/or negative control(s). The kitmay be packaged in any suitable manner, typically with the elements in asingle container or various containers as necessary.

RNA and Protein Detection and Quantification Assays

In another embodiment, the present invention relates methods fordetecting or quantifying mRNA or protein in a test sample obtained fromone or more subjects. The information obtained by detecting orquantifying mRNA or protein in a test sample obtained from a subject canbe used in a variety of different ways. For example, the presence,absence or amount of mRNA or protein detected or quantified in subjectscan be used to diagnose those subjects suffering from major depressionor a related disorder or at risk of developing major depression or arelated disorder, provide a prognosis for or predict or diagnose aresponse to treatment for a subject suffering from major depression or arelated disorder or identifying subjects for selection or inclusion in aclinical trial for treating major depression or a related disorder.Additionally, the presence, absence or amount of mRNA or protein can beused to analyze the results of a clinical trial for subjects beingtreated for major depression or a related disorder. Specifically, therelationship between the presence, absence or amount of the mRNA orprotein detected or quantified in the test samples and the clinicaloutcome of said subjects can be determined.

The methods described herein involve obtaining a test sample from saidsubject(s). The subject may or may not be experiencing any symptoms ofmajor depression or a related disorder at the time the test sample isobtained. In this embodiment, a test sample is any biological samplewhich contains the RNA or protein of the subject. Test samples can beprepared using techniques well known to those skilled in the art such asby obtaining a specimen from an individual and, if necessary, disruptingany cells contained therein to release RNA or protein. Examples of testsamples include, but are not limited to, whole blood, serum, plasma,cerebrospinal fluid, sputum, bronchial washing, bronchial aspires,urine, lymph fluids, and various external secretions of the respiratory,intestinal and genitourinary tracts, tears, saliva, milk, white bloodcells, myelomas and the like, etc.

Once the test sample(s) is obtained, it is analyzed, using routinetechniques known in the art, in order to determine or quantify thepresence, absence or amount of: (a) at least one mRNA which comprisesnucleotides 1 to 316 of SEQ ID NO:2; (b) at least one mRNA transcribedfrom SEQ ID NO:1; (c) at least one protein having an amino acid sequenceof SEQ ID NO:3 or SEQ ID NO:4; (d) at least one polypeptide translatedfrom SEQ ID NO:1; or (e) any combinations of (a)-(d). Additionally, thetest sample may optionally be further analyzed for the presence, absenceor amount of mRNA transcribed from the HTR1A gene or a polypeptidetranslated from the HTR1A gene.

As discussed above, once a test sample is obtained, it can be analyzed,using routine techniques known in the art for the presence, absence oramount of at least one mRNA transcribed from SEQ ID NO:1. Examples ofmRNAs transcribed from SEQ ID NO:1 include, but are not limited to, SEQID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:33. Alternatively, thetest sample can be analyzed for the presence, absence or amount of atleast one polypeptide translated from SEQ ID NO:1. Examples ofpolypeptides translated from SEQ ID NO:1 include, but are not limitedto, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32 and SEQ ID NO:34.

Once the presence, absence or amount of a mRNA or a protein as specifiedin (a)-(e) above has been determined or quantified in a test sample,then further determinations can be made, such as, diagnosing whether thesubject has major depression or a related disorder or is at risk ofdeveloping major depression or a related disorder, providing a prognosisfor or predicting the response to treatment for a subject having majordepression or a related disorder, determining whether the subject shouldbe selected for inclusion in a clinical trial for treatment of majordepression or a related disorder, or analyzing the relationship betweenthe frequency of presence or relative amounts of at least one mRNA orpolypeptide in subjects, and their clinical outcome. Additionally, ifthe test sample is further analyzed for the presence, absence or amountof mRNA transcribed from the HTR1A gene or a polypeptide translated fromthe HTR1A gene, the information pertaining to mRNA(s) or polypeptide(s)transcribed or translated from DEP2 may be used in combination with theinformation pertaining to mRNA(s) or polypeptide(s) transcribed ortranslated from HTR1A to make further determinations, as elaboratedabove.

For example, a test sample can be obtained from a subject. The testsample can then be analyzed using routine techniques known in the art,in order to determine or quantify the presence, absence or amount of (a)at least one mRNA which comprises nucleotides 1 to 316 of SEQ ID NO:2;(b) at least one mRNA transcribed from SEQ ID NO:1; (c) at least oneprotein having an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4; (d)at least one polypeptide translated from SEQ ID NO:1; or (e) anycombinations of (a)-(d). If, for example, the presence of (a) at leastone mRNA which comprises nucleotides 1 to 316 of SEQ ID NO:2; (b) atleast one mRNA transcribed from SEQ ID NO:1; (c) at least one proteinhaving an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4; (d) atleast one polypeptide translated from SEQ ID NO:1; or (e) anycombinations of (a)-(d) is detected, then a diagnosis can be made forsaid subject related to major depression or a related disorder orrelated to risk of developing major depression or a related disorder.This information can also be useful for providing a prognosis for orpredicting the response to treatment for a subject already diagnosed assuffering from major depression or a related disorder. Moreover, thisinformation can be used to determine whether or not the subject shouldor could be selected for inclusion in a clinical trial for treatment ofmajor depression or a related disorder. Further, the frequency ofpresence of: (a) at least one mRNA which comprises nucleotides 1 to 316of SEQ ID NO:2; (b) at least one mRNA transcribed from SEQ ID NO:1; (c)at least one protein having an amino acid sequence of SEQ ID NO:3 or SEQID NO:4; (d) at least one polypeptide translated from SEQ ID NO:1; or(e) any combinations of (a)-(d) can be used to analyze the results of aclinical trial for subjects being treated for major depression or arelated disorder. Specifically, the relationship between the presence ofsaid mRNA, protein, polypeptide or combinations thereof in the testsamples and the clinical outcome of said subjects can be determined.Similarly, any of the above further determinations might be made on thebasis of absence of: (a) at least one mRNA which comprises nucleotides 1to 316 of SEQ ID NO:2; (b) at least one mRNA transcribed from SEQ IDNO:1; (c) at least one protein having an amino acid sequence of SEQ IDNO:3 or SEQ ID NO:4; (d) at least one polypeptide translated from SEQ IDNO:1; or (e) any combinations of (a)-(d), or on the basis of detectionor quantification that an amount of: (a) at least one mRNA whichcomprises nucleotides 1 to 316 of SEQ ID NO:2; (b) at least one mRNAtranscribed from SEQ ID NO:1; (c) at least one protein having an aminoacid sequence of SEQ ID NO:3 or SEQ ID NO:4; (d) at least onepolypeptide translated from SEQ ID NO:1; or (e) any combinations of(a)-(d), is within a certain range.

Techniques for identifying the presence, absence or amount of mRNAs orproteins in a test sample are well known in the art. For example,techniques for identifying the presence, absence or amount of mRNAsinclude, but are not limited to, reverse transcriptase, cDNAmicroarrays, quantitative PCR and Northern blotting. Techniques foridentifying the presence, absence or amount of proteins include, but arenot limited to, ELISA, RIA, Western blotting, fluorescence activatedcell sorting and immunohistochemical analysis. These techniques will bediscussed briefly below.

RNA Techniques

Reverse transcriptase can be used to prepare a cDNA by used of an oligodT primer which is annealed to the poly A sequence of the RNA. Examplesof reverse transcriptases that can be used include, but are not limitedto, ImProm-II Reverse Transcriptase (Promega, Madison, Wis.) and BDPowerscript Reverse Transcriptase (BD Biosciences, Franklin Lakes,N.J.). Methods for using reverse transcriptases to prepare and obtaincDNA molecules are well known in the art and are described in Sambrook,J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1989).

A cDNA microarray is an array of multiple cDNA molecules, fixed inaddressable locations (such as on a chip assay), to which complementarynucleic acids in applied samples may hybridize (see Hegde et al.,Biotechniques 29(3):548-562 (2000)). cDNA microarrays provide forqualitative and quantitative analysis of mRNA expression of themolecules contained in the array.

Quantitative PCR allows for the direct monitoring of the progress of aPCR amplification as it is occurring, without the need for repeatedsampling of the reaction products. In quantitative PCR, the reactionproducts may be monitored as they are generated and are tracked afterthey rise above background but before the reaction reaches a plateau.The number of cycles required to achieve a chosen level of fluorescencevaries directly with the concentration of amplifiable targets at thebeginning of the PCR process, enabling a measure of signal intensity toprovide a measure of the amount of target DNA in a sample in real time.Quantitative PCR according to the present invention may be performed onany suitable instrument, including, but not limited to, Mx4000 orMx3000P (Stratagene, La Jolla, Calif.), ABI7700 or ABI7000 (AppliedBioSystems Inc., Foster City, Calif.), MJ Opticon (MJ Research,Watertown, Mass.), iCycler (Bio-Rad, Hercules, Calif.), RotorGene 3000(Corbett Life Sciences, Mortlake, NSW, Australia), and the SmartCycler(Cepheid, Sunnyvale, Calif.).

In solid phase detection, hybridization of a probe to the sequence ofinterest, such as an RNA, is detected directly by visualizing a boundprobe by using Northern blotting. In this technique, RNA is isolatedfrom a subject. The RNA is then cleaved with a series of restrictionenzymes that cleave infrequently in the genome and not near any of themarkers being assayed. The RNA is then separated (such as, but notlimited to, by agarose gel electrophoresis) and transferred to amembrane. At least one probe which has been labeled with, for example, aradioactive, fluorescent or enzymatic label, specific for thepolymorphism being detected is allowed to contact the membrane under acondition of low, medium, or high stringency conditions. Unbound probeis removed and the presence of binding is detected by visualizing thelabeled probe.

Protein Techniques

ELISA involves the fixation of a test sample containing a proteinsubstrate of interest to a surface such as a well of a microliter plate.A substrate specific antibody coupled to an enzyme is applied andallowed to bind to the substrate. Presence of the antibody is thendetected and quantitated by a colormetric reaction employing the enzymecoupled to the antibody. Enzymes commonly in ELISAs include, but are notlimited to, horseradish peroxidase and alkaline phosphatase. If wellcalibrated and within the linear range of response, the amount ofsubstrate present in the sample is proportional to the amount of colorproduced. A substrate standard is generally employed to improvequantitative accuracy.

Another technique that can be used is a radioimmunoassay (“RIA”). Oneversion of RIA involves the precipitation of a desired substrate (suchas a protein of interest) with a specific antibody and detectablylabeled antibody binding protein (the antibody binding protein can belabeled with any detectable isotope known in the art) immobilized on aprecipitable carrier, such as, but not limited to, agarose beads. Thenumber of counts in the precipitated pellet is proportional to theamount of substrate present in the test sample. In an alternate versionof RIA, a labeled substrate (such as a protein of interest) and anunlabelled antibody binding protein are employed. A test samplecontaining an unknown amount of substrate is added in varying amounts.The decrease in precipitated counts from the labeled substrate isproportional to the amount of substrate in the added sample.

Western blot involves separation of a substrate (such as a protein ofinterest) from another protein by means of an acrylamide gel followed bytransfer of the substrate to a membrane (such as, but not limited to,nylon or PVDF). The presence of the substrate is then detected byantibodies specific to the substrate. The antibodies are then detectedby antibody binding reagents. Antibody binding reagents may include, butare not limited to, protein A or other antibodies. The Antibody bindingreagents may labeled with a detectable label as described previouslyherein. Detection may be by autoradiography, colorimetric reaction orchemiluminescence. Western blotting allows for both the quantitation ofan amount of substrate and a determination of the substrate's identityby a relative position on the membrane which is indicative of amigration distance in the acrylamide gel during electrophoresis.

Fluorescence activated cell sorting (“FACS”) involves detection of asubstrate (such as a protein of interest) in situ in cells by substratespecific antibodies. The substrate specific antibodies are linked tofluorophores. Detection is by means of a cell sorting machine whichreads the wavelength of light emitted from each cell as it passesthrough a light beam. This method may employ two or more antibodiessimultaneously.

Immunohistochemical analysis involves detection of a substrate (such asa protein of interest) in situ in fixed cells by substrate specificantibodies. The substrate specific antibodies may be enzyme linked orlinked to fluorophores. Detection is by microscopy and subjectiveevaluation. If enzyme linked antibodies are employed, a calorimetricreaction may be required.

Kits

The present invention also provides kits that enable or allow for thedetection or quantification of (a) at least one mRNA which comprisesnucleotides 1 to 316 of SEQ ID NO:2; (b) at least one mRNA transcribedfrom SEQ ID NO:1; (c) at least one protein having an amino acid sequenceof SEQ ID NO:3 or SEQ ID NO:4; (d) at least one polypeptide translatedfrom SEQ ID NO:1; or (e) any combinations of (a)-(d) in one moresubjects. These kits are useful for diagnosing those subjects sufferingfrom major depression or a related disorder or at risk of developingmajor depression or a related disorder, providing a prognosis for orpredicting a response to treatment for a subject suffering from majordepression or a related disorder, identifying subjects for selection orinclusion in a clinical trial for treating major depression or a relateddisorder, or for analyzing the results of a clinical trial for treatingmajor depression or a related disorder relationship.

The kits can be produced in a variety of ways. For example, the kitscontain at least one reagent useful for detecting or quantifying thepresence, absence or amount of: (a) at least one mRNA which comprisesnucleotides 1 to 316 of SEQ ID NO:2; (b) at least one mRNA transcribedfrom SEQ ID NO:1; (c) at least one protein having an amino acid sequenceof SEQ ID NO:3 or SEQ ID NO:4; (d) at least one polypeptide translatedfrom SEQ ID NO:1; or (e) any combinations of (a)-(d). Additionally, anyof the kits described above in (a)-(e) can further contain at least onereagent useful for detecting or quantifying the presence, absence oramount of the presence, absence or amount of mRNA transcribed from theHTR1A gene or a polypeptide translated from the HTR1A gene. Examples ofthe at least one reagent that can be included in the kits describedherein are a reverse transcriptase, one or more primers for amplifyingcDNA or at least one antibody. In addition, the kits can further contain(a) instructions describing how to detect or quantify the presence,absence or amount of at least one mRNA or at least one protein in a testsample; (b) ancillary reagents such as buffering agents, nucleic acidstabilizing reagents, protein stabilizing reagents, and signal producingsystems (such as, but not limited to, fluorescence generating systems);or (c) positive and/or negative control(s). The kit may be packaged inany suitable manner, typically with the elements in a single containeror various containers as necessary.

Screening Assays and Methods of Treatment

In another embodiment, the present invention relates to methods (alsoreferred to herein as “screening assays” or “screening methods”) foridentifying compositions, namely candidate or test compounds or agents(such as, but not limited to, small molecules, antibodies, nucleicacids, peptides, peptidomimetics, or other drugs), which: (a) bind to aprotein translated from SEQ ID NO:1; (b) modulate the activity orexpression of a protein translated from SEQ ID NO:1 (such as byinhibiting, reducing or decreasing the activity, reducing or decreasingthe expression, or by stimulating or increasing the activity, orstimulating or increasing the expression, of the protein); or (c)modulate the expression of an mRNA molecule transcribed from SEQ ID NO:1(such as by reducing or decreasing the expression or by stimulating orincreasing the expression.

Since genetic linkage between DEP2 and major depressive disorder hasbeen established, it is thought that compositions identified pursuant tothe screening methods described herein may be useful in treating majordepression or a related disorder.

Examples of proteins translated from SEQ ID NO:1 include, but are notlimited to, (i) Lhpp (SEQ ID NO:10), (ii) naturally occurring proteinvariants of Lhpp (SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO; 17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:29); (iii)Dep2-1a (SEQ ID NO:3); (iv) Dep2-1b (SEQ ID NO:4); Dep2-2 (SEQ IDNO:27); (v) Dep2-4 (SEQ ID NO:32); and (vi) Dep2-5 (SEQ ID NO:34).Examples of RNA molecules transcribed from SEQ ID NO:1 include, but arenot limited to, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:33.

As will be discussed in more detail herein, the present inventionincludes a method of determining whether a composition, identified inaccordance with the methods described herein, is a potential therapy formajor depression or a related disorder by initially administering thecomposition to a mammal (for example, an animal model). One may thenmonitor for major depression-related symptoms of the animal or the levelor activity of a protein translated from SEQ ID NO:1 in the testsubject. A decrease in the appearance of such symptoms indicates thepotential suitability of the composition of interest in the treatment ofmajor depression or related disorders. Such a finding in an animal modelwould then lead to use of the composition in human clinical trials.

Suitable animal models for such experiments include, but are not limitedto, behavioral despair or mouse forced swim test (Arch. Int.Pharmacodyn. 229:327-336 (1977), Psychopharmacology 94:147-160 (1988));tail suspension test (Psychopharmacology 85:367-370 (1985)); elevatedplus maze test (Psychopharmacology 92:180-185 (1987)); open field test(Behav. Brain Res. 134:49-57 (2002)); dark-light transitions test(Pharmacol. Biochem. Behav. 15:695-699 (1981)); Irwin test (Brain Res.Vol. 835:18-26 (1999); Psychopharmacology 147:2-4 (1999)); inescapablestress test (learned helplessness) (Seligman and Maier, J. Exp. Psychol74:1-9, (1967)); chronic mild stress (Ducottet et al., Prog.Neuro-Psychopharmacol. Biol. Psychiatry 27:625-631 (2003); Kopp et al.,Behav. Pharmacol. 10:73-83 (1989)); and novelty-suppressed feeding model(Bodnoff et al., Psychopharamcology 97:277-279 (1989)).

The present invention additionally relates to the compositionsidentified by use of the above screening methods as well as to methodsof using these compositions in the treatment of major depression or arelated disorder. More specifically, once a composition of interest hasbeen identified, the composition may be used in clinical trials todetermine whether it actually alleviates the symptoms of majordepression or a related disorder or at least decreases the severitythereof.

Also, it is submitted that the proteins described herein may be used tocharacterize the physical properties of compositions which may be usedto ultimately treat major depression or a related disorder and thus inthe “design” of such compositions. Thus, based upon such properties, onemay design a composition or compound that has the ability to have asignificant degree of binding affinity to a protein translated from SEQID NO:1, thereby modulating the activity of the protein. Such acomposition or compound could then be used in the treatment of majordepression or a related disorder.

Furthermore, one may detect binding of a test composition to a proteintranslated from SEQ ID NO:1 by subjecting the protein to, for example,nuclear magnetic resonance (“NMR”) alone and in the presence of thecomposition.

Characteristic changes in the NMR spectrum of the protein may then allowone to determine whether and how the composition has bound to theprotein. This procedure may be repeated for a series of compounds,enabling discovery of relationships between compound structure andbinding to the target protein. This iterative process is known as“structure-activity relationships by NMR” or “SAR by NMR” (Shuker etal., Science 274:1531-1534 (1996); SAR by NMR is described in U.S. Pat.Nos. 5,891,643, 5,989,827, 5,804,390, 6,043,024 and 6,897,337).

Similarly, one may identify the structure of a composition bound to theprotein by x-ray diffraction techniques. By iterative operation of thistechnique, one may optimize lead compositions or compounds so as todevelop the most efficacious therapeutic compositions or compounds forthe treatment of major depression or a related disorder.

One method of identifying compositions that modulate the amount oractivity of a protein translated from SEQ ID NO:1 or that modulate theexpression of an mRNA molecule that is transcribed from SEQ ID NO:1 is areporter gene assay. It is well known to those skilled in the art that areporter gene assay may be carried out in an intact cell transfectedwith the reporter gene construct, in extracts from a cell transfectedwith the reporter gene construct, or in a cellular extract (for example,reticulocyte lysate) to which the reporter gene construct is added. Itis further recognized that reporter gene assays may be carried out usingcells or extracts that naturally contain a protein translated from SEQID NO:1 or an mRNA molecule transcribed from SEQ ID NO:1, cells intowhich a vector for the expression of a protein translated from SEQ IDNO:1 or an mRNA molecule transcribed from SEQ ID NO:1 that has beentransfected (transiently or stably), or extracts to which a purified orpartially purified amino acid translated from SEQ ID NO:1 or an mRNAmolecule transcribed from SEQ ID NO:1 is added. In the presentinvention, it is preferred that a protein translated from SEQ ID NO:1 oran mRNA molecule transcribed from SEQ ID NO:1 be purified from a humancell or tissue, or from expression in an heterologous system. Further,it is also well known in the field that reporter gene assays may beconducted in cells or extracts that are of human origin, or that comefrom a different mammal or organism. It is additionally recognized thatthere are many regulatory sequences (such as promoters) that can be usedto initiate transcription in a reporter gene construct, and that thechoice of a regulatory sequence may be determined more by the particularcell or extract in which the assay will be conducted. It is stillfurther well known that there are a variety of reporter genes that areamenable to screening assays, including high throughput screeningassays. Examples of reporter genes include those which are themselvesfluorescent, luminescent or have easily detected spectralcharacteristics (for example, a green fluorescent protein), as well asthose having well-characterized fluorescent, luminescent or colorimetricsubstrates (for example, beta-galactosidase, luciferases). It is finallyrecognized that certain cofactors may be added as purified or partiallypurified components to a reporter gene assay. A discussion of reportersystems can be found in Current Protocols in Pharmacology (2003), Units6.2.1-6.2.11, Wiley & Sons, Inc.

An additional embodiment of reporter gene assays involve the use of atleast one substrate for a protein translated from SEQ ID NO: 1. Thesubstrate can be added before the protein is exposed to the testcomposition or simultaneously with the test composition, provided thatthe protein is exposed to the substrate for a time and under conditionssufficient to allow the protein to react with the substrate in order toproduce a reaction product. Any substrate wherein the phosphorylation ofsaid substrate is capable of being modified by a protein translated fromSEQ ID NO:1 can be used in the reporter gene assays described herein.Proteins translated from SEQ ID NO:1 that can be used to modify thephosphorylation of said substrate, include, for example, proteins havingan amino acid sequence selected from the group consisting of: SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:27. Examples ofsubstrates that can be used include, but are not limited to,phosphohistidine, phospholysine, phosphodiimide, pyrophosphate or anypeptide or protein that is phosphorylated on a histidine or a lysine.For example, a reporter gene assay for screening a composition for theability to inhibit activity of SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 andSEQ ID NO:27 can be performed. The method involves exposing a protein toa test composition and then measuring the presence or absence of areaction product or complex. The lack of a reaction product or complexindicates that the composition has the ability to inhibit the activityof the protein. Prior to exposing the protein to the test composition, asubstrate can be added to the protein. Alternatively, the substrate canbe added when the protein is exposed to the test composition.

An additional substrate that can be used is a radioactive enzymesubstrate. In such an embodiment, the reporter gene encodes an enzyme(for example, chloramphenicol acetyltransferase) having a substrate thatis readily separated from the corresponding reaction product. This typeof radioactive detection assay may be utilized in order to identify acompound that binds to or modulates a protein translated from SEQ IDNO:1 or that modulates the expression of an mRNA molecule transcribedfrom SEQ ID NO:1. It is well known to those skilled in the art that theseparation and detection of radioactive compounds may be accomplished bya variety of chromatographic and other methods. A discussion ofradioactive reporter gene assays can be found in Current Protocols inPharmacology (2003), Units 6.4.1-6.4.11, Wiley & Sons.

An additional assay that may be used to detect a composition or compoundhaving the ability to modulate the activity or expression of a proteintranslated from SEQ ID NO:1 or modulate the expression of an mRNAmolecule transcribed from SEQ ID NO:1 is the scintillation proximityassay. This assay is based upon the binding of a radiolabeled tracer toa protein translated from SEQ ID NO:1 or an mRNA molecule transcribedfrom SEQ ID NO:1 that has been exposed to the composition or compound ofinterest. The scintillant is incorporated into small fluoromicrospheresto which target macromolecules (for example, proteins or mRNAs) attach.If a radioactive molecule (for example, ³H) binds to the target, it isbrought close enough to the bead to stimulate the scintillant to producelight. On the other hand, unbound radioactivity is not detected if thebead is outside the distance subatomic particles produced by the decayare likely to travel. Thus compositions or compounds that bind to aprotein translated from SEQ ID NO:1 or an mRNA molecule transcribed fromSEQ ID NO:1 may be detected by changes in the amount ofscintillant-emitted light. A discussion of scintillation proximityassays can be found in Current Protocols in Pharmacology (2003), Unit9.4.9-9.4.10, Wiley & Sons, Inc.

Another assay which may be utilized in the identification ofcompositions that affect the binding or that modulate a proteintranslated from SEQ ID NO:1 to mRNA is a filter binding assay. Anexample of the filter binding assay that may be utilized for a proteintranslated from SEQ ID NO:1 involves immobilization of an RNA molecule(for example, all or part of an mRNA transcribed from SEQ ID NO:1) on asolid support, exposure of the immobilized RNA to a protein translatedfrom SEQ ID NO:1 in the absence or presence of compositions thought tobind or inhibit protein translated from SEQ ID NO:1, and quantitation ofa protein translated from SEQ ID NO:1 on the solid support. It is wellknown to those skilled in the art that the solid support may be anitrocellulose or other filter, or any of a variety of beads ormicroparticles. It is further recognized that a protein translated fromSEQ ID NO:1 used in the assay may be purified from an heterologousexpression system, and will advantageously be tagged such that it can bedetected using commonly available reagents. For example, a proteintranslated from SEQ ID NO:1 may be a fusion to a ‘tag’ sequenceexpressed in E. coli (Tateiwa et al., Journal of Neuroimmunology120:161-69 (2001)). Compositions that bind to or inhibit a proteintranslated from SEQ ID NO:1 may be identified by a increase or reductionin the amount of a protein translated from SEQ ID NO:1 on the solidsupport, relative to a reaction in which no test compound was added.

Another type of assay that may be useful to screen for compositions thatbind to protein translated from SEQ ID NO:1 is a fluorescencepolarization assay. This method detects molecular interactions and isbased on the concept that fluorescent molecules excited by lightpolarized in one plane will emit a fluorescent signal again in apolarized manner. The rotational relaxation time is proportional to themolecular volume if other physical variables are unchanged. Thus, whenbinding to a larger molecule restricting rotation and tumbling, theemission remains polarized, such polarization can be calculated and isdirectly proportional to the fraction of bound ligand. Change influorescence polarization thus accounts for the ratio of bound versustotal ligand. For a protein translated from SEQ ID NO:1, one embodimentof a fluorescent polarization assay would involve a fluorescentlylabeled polynucleotide comprising all or part of a nucleic acid of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:33.Compositions that bind to a protein translated from SEQ ID NO:1 may bedetected by a reduction of fluorescent polarization attributable to thelabeled polynucleotide. A discussion of fluorescent polarization assayscan be found in Current Protocols in Pharmacology, (2003), Units9.4.12-9.4.13 Wiley & Sons, Inc.

Another type of assay that may be used to screen for compositions thatbind to a protein translated from SEQ ID NO:1 is the spin-screeningassay. This method detects molecular interactions, and is based on theconcept that the sedimentation rate of molecules or molecular complexesin solution depends on mass and shape. In particular, the sedimentationrate of a small molecule alone is expected to be substantially differentfrom that of the same small molecule bound to a macromolecule. Thus,when a directional force is applied (for example, by spinning a solutionat high speed in a centrifuge), small molecules that bind to a proteintranslated from SEQ ID NO:1 can be readily separated from other smallmolecules in a mixture that do not. Separation of bound from unboundsmall molecules can also be accomplished by including a size exclusionfilter within the centrifuge tube, such that unbound small moleculespass through the filter but bound small molecules do not. It isrecognized by those skilled in the art that molecules separated in thisfashion can be identified by a variety of spectroscopic and othermethods. In one embodiment, a spin-screening assay includes detection bymass spectrometry.

In another embodiment, the present invention relates to methods ofdetermining the in vivo activity of a composition identified as apotential therapy for the treatment of major depression or a relateddisorder. These methods involve obtaining at least two (2) test samplesfrom a subject, preferably a human, being treated for major depressionor a related disorder. A first test sample can be considered to be atest sample that is obtained at a period in time before the subject hasbegun a course of treatment with the test composition. Alternatively, afirst test sample can also be considered to be a test sample obtained ata period in time during which a subject has been receiving a course oftreatment with the test composition. A second test sample can beconsidered to be a test sample that is obtained at a period in time thatis subsequent to the obtaining of the first test sample. For example,the second test sample can be obtained after a period of time haselapsed after the subject has begun an initial course of treatment withthe test composition (meaning that the subject had not previouslyreceived the test composition prior obtaining the first test sample).Alternatively, if a subject has been receiving treatment with a testcomposition, a first test sample can be obtained from said subject.After a period of time has elapsed (for example, three (3) months)during which said subject is still being treated with the testcomposition, a second test sample can be obtained from said subject. Adiscussion of what constitutes a test sample and examples of testsamples has already been provided previously herein and is incorporatedherein by reference.

Once the test samples are obtained, they are analyzed, using routinetechniques known in the art (which have been discussed previouslyherein), to determine or quantify the: (a) amount or activity of aprotein translated from SEQ ID NO:1; or (b) amount of mRNA transcribedfrom SEQ ID NO:1, in each of the test samples. The amount or activity ofa protein translated from SEQ ID NO:1 or the amount of mRNA transcribedfrom SEQ ID NO:1 that was detected or quantified in each of the testsamples is compared. If, for example, the amount or activity of proteinor the amount of mRNA determined or quantified in the second test sample(which was the test sample obtained after the subject began a course oftreatment with the composition) is the same (namely, equal) as theamount or activity of protein or the amount or activity of mRNAdetermined or quantified in the first test sample (which was the testsample obtained from the subject prior to undergoing said course oftreatment with the composition), this indicates that the compositionlacks therapeutic activity. In contrast, if the amount or activity ofthe protein or the amount of mRNA determined or quantified in the secondtest sample has changed, namely, has increased or decreased, whencompared to the amount or activity of the protein or the amount of mRNAdetermined or quantified in the first test sample, this indicates thatthe composition possesses some type or degree of therapeutic activity.

In addition, in yet another embodiment, the present invention relates tomethods for determining the presence or absence of activity of acomposition identified pursuant to the screening methods describedherein that is being used to treat a subject suffering from majordepression or a related disorder. The method involves observing thephenotype of said subject prior to the subject being administered thetest composition (“first visit”). For example, observation of thesubject's phenotype should be based on a method that has been validatedas a measure of major depression or a related disorder. Such validatedmethods include, but are not limited to: the Hamilton depression ratingscale (Hamilton, J. Neurol. Neurolsurg. Psychiatry 23:56-62 (1960),Schedule for affective disorders and schizophrenia (Spitzer andEndicott, Schedule for affective disorders and schizophrenia, lifetimeversion. New York, N.Y.: New York State Psychiatric Institute,Biometrics Research. 1975), Montgomery-Asberg depression rating score(Montgomery, Br. J. Psychiatry 134:382-389 (1979)) and the Structuredclinical interview for DSM-IV (First et al., Structured ClinicalInterview for DSM-IV. Washington, D.C.: American Psychiatric Press1997). After observation, the subject is administered the testcomposition for a time and under conditions that are sufficient for thecomposition to either: (a) bind to, inhibit, increase, decrease orreduce the amount of a protein translated from SEQ ID NO:1; or (b)increase or reduce the amount of a mRNA molecule transcribed from SEQ IDNO:1. After the subject has been administered the test composition for atime and under the conditions described above, the phenotype of thesubject is again observed, preferably, using the same validated methodas was used to establish the initial phenotype. Observable improvementin the phenotype of the subject at the second observation compared tothe first observation indicates that the composition has some type ordegree of therapeutic activity. A lack of observable differences in thephenotype of the subject at the first observation compared to the secondobservation indicates that the composition does not possess therapeuticactivity. The steps of observing the phenotype of the subject andadministering the composition to said subject can be repeated for aslong as the treating physician deems necessary. The physician may thencompare the phenotype of the subject between any pair of observations tojudge whether the composition has some type or degree of therapeuticactivity. In one aspect of this embodiment, commonly used in clinicalresearch, the phenotype observed at the time of the last administrationof the test composition is referred to as the “last visit”. Eventually,the phenotype of the subject prior to initiation of treatment iscompared with the phenotype of the subject at the last visit. Observabledifferences in the phenotype of the subject prior to initiation oftreatment compared to the last visit indicates that the compositionpossesses some type or degree of therapeutic activity. A lack ofobservable differences in phenotype of the subject prior to initiationof treatment compared to the last visit indicates that the compositiondoes not possess any type of therapeutic activity.

In another embodiment, the present invention relates to the compositionsidentified by methods described herein in the prevention of majordepression or a related disorder or the treatment of major depression ora related disorder. More specifically, the present inventioncontemplates a method for at least substantially preventing in a subjectmajor depression or a related disorder by administering to a subject inneed of treatment thereof, a therapeutically effective amount of atleast one composition that has been identified by the hereinbeforedescribed methods that: (a) modulates the activity of a proteintranslated from SEQ ID NO:1; (b) reduces the amount of a proteintranslated from SEQ ID NO:1; (c) increases the amount of a proteintranslated from SEQ ID NO:1; or (d) modulates the level of expression ofan mRNA molecule transcribed from SEQ ID NO:1. Administration of aprophylactic composition can occur prior to the manifestation ofsymptoms characteristic of major depression or a related disorder.

Additionally, the present invention further contemplates a method oftreating a subject suffering from major depression or a related disorderby administering to a subject in need of treatment thereof, atherapeutically effective amount of at least one composition that hasbeen identified by the hereinbefore described methods that: (a)modulates the activity of a protein translated from SEQ ID NO:1; (b)reduces the amount of a protein translated from SEQ ID NO:1; (c)increases the amount of a protein translated from SEQ ID NO:1; or (d)modulates the level of expression of an mRNA molecule transcribed fromSEQ ID NO:1.

By way of example, and not of limitation, examples of the presentinvention shall now be given.

Example 1 Identification of Genetic Linkage and Association Between DEP2and Major Depressive Disorder

Genetic linkage between DEP2 and major depressive disorder wasestablished in a pedigree-based study in the Mormon population of Utah.The ascertainment and characteristics of a majority of these pedigreeshas been described (Abkevich et al., Am. J. Hum. Genet. 73:1271-1281(2003)). In the study described herein, a total of 93 pedigrees thatcontain a minimum of four females affected with major depressivedisorder (DSM-IV-TR sections 296.2x or 296.3x) were selected for geneticanalysis. These pedigrees comprised 744 affected females.

Affected individuals were genotyped and genome-wide linkage analysis wasperformed as described (Abkevich et al., op. cit.). Two meaningfuldifferences between the present study and our previously published workare: first, that additional pedigrees were ascertained; second, that thedefinition of affected status was different as it did not includebipolar disorder in this study.

Using a dominant genetic model and considering only females with majordepressive disorder as affected, evidence of linkage on chromosome 10 atmarker D1051676 was observed (heterogeneity LOD score (HLOD) 2.4). Upongenotyping of additional markers in the 26 centimorgan (“cM”) intervalbetween D1052322 and D1051700, the linkage evidence increased to a peakHLOD of 3.4 at D105214 (FIG. 27 and Table 1).

The serotonin receptor 1A (Htr1a) is a therapeutic target in themanagement of depressive and anxiety disorders (Barnes and Sharp,Neuropharmacology 38:1083-1152 (1999)). A common polymorphic site in thecorresponding gene (HTR1A) has been described, such that the 1019^(th)nucleotide upstream of the transcriptional start site naturally occursas either cytosine or guanosine (Wu and Comings, Psych. Genet. 9:105-106(1999)). Results of in vitro experiments suggest that the variant allele(-1019G) prevents binding of a transcriptional repressor, resulting inenhanced Htr1a expression (Lemonde et al., J. Neurosci., 23:8788-8799(2003)). Either the -1019G allele or homozygous -1019GG genotype hasbeen associated with depression, suicide, bipolar disorder, panicdisorder with agoraphobia, neuroticism and decreased anti-depressantresponse (Arias et al., Mol. Psych. 7:930-932 (2002); Strobel et al., J.Neural Transm., 110-1445-1453 (2003); Lemonde et al., J. Neurosci.,23:8788-8799 (2003); Rothe et al., Int. J. Neuropsychopharmacol.7:189-192 (2004); Huang et al., Int. J. Neuropsychopharmacol. 7:441-451(2004); Serretti et al., Int. J. Neuropsychopharmacol. 7:453-460 (2004);Lemonde et al., Int. J. Neuropsychopharmacol. 7:501-506 (2004); Arias etal., J. Psychopharmacol. 19:166-172 (2005)).

In the Utah population, HTR1A allele -1019G and genotype -1019GG were1.1- and 1.3-fold over-represented among individuals affected with majordepressive disorder compared to unaffected individuals (one-tailedp=0.05 and 0.02, respectively). Hence, linkage analysis was stratifiedaccording to HTR1A −1019 alleles. That is, only individuals with majordepressive disorder, and also carrying one or two copies of theHTR1A-1019G risk allele, were considered affected. In a genome-wideHTR1A-conditional linkage analysis using a dominant genetic model andalso restricted to female sex, the observed evidence of linkage onchromosome 10 strengthened to a peak HLOD of 3.1 at D10S1222. Uponinclusion of additional marker data in the 26 cM interval betweenD10S2322 and D10S1700, the linkage evidence increased to a peak HLOD of4.4 at D10S575 (FIG. 27 and Table 1).

The conditional linkage method improved upon the previously performedtraditional linkage analysis in three ways. First, as noted above, itrevealed stronger evidence supporting linkage of a dominant gene tomajor depressive disorder in females on chromosome 10 in the vicinity ofD105575. Second, it narrowed the linkage region (as defined by a drop ofHLOD of either 1 or 2 from the peak value), such that the location ofthe linked gene was better defined. Third, and most importantly, itrevealed linkage evidence in a distinct subset of pedigrees. Furtherinvestigation of those pedigrees was crucial to the discovery of DEP2 asa gene linked to major depressive disorder.

As a next step to identify a gene linked to major depressive disorder,each gene in the conditional linkage region was resequenced inrepresentative affected females from each of sixteen pedigrees. Thesepedigrees were selected on the basis of having a familial HLOD of atleast 0.4. Among these pedigrees, six had not shown linkage evidencewithout stratification on the basis of HTR1A alleles. The frequencies ofvariant alleles among the 22 chromosomes that segregated with majordepressive disorder within these pedigrees was compared to thefrequencies among 60 control chromosomes. For seven single nucleotidepolymorphisms (“SNPs”) within SEQ ID NO:1, statistically significantfrequency differences were observed. Additionally, a statistical trendwas observed for an eighth SNP in SEQ ID NO:1 (Tables 2 and 3). Twopairs of these SNPs (DEP2.0001 and DEP2.0002, DEP2.0004 and DEP2.0005)were in complete linkage disequilibrium with each other. Between thesemarkers, only DEP2.0002 and DEP2.0004 are described further. One SNP ineach of six other genes in the linkage region showed statisticallysignificant frequency differences between the 22 chromosomes thatsegregated with major depressive disorder within these pedigrees and theset of 60 control chromosomes (Table 3).

For three of six tested SNPs in SEQ ID NO:1, statistically significantfrequency differences were also observed between the 22 chromosomes thatsegregated with major depressive disorder and an independent set of 180control chromosomes (Table 4). None of the six tested SNPs from othergenes showed statistical significance in this test. For five of the sixtested SNPs in SEQ ID NO:1, statistically significant frequencydifferences were also observed between the 22 chromosomes thatsegregated with major depressive disorder and a third independent set of708 control chromosomes (Table 5).

To confirm the relationship between DEP2 genotypes and major depressivedisorder, genetic association studies comparing genotype frequenciesbetween individuals affected with major depressive disorder (notascertained on the basis of familial history of disease) and healthycontrols were performed in two populations. Consistent with the dominantlinkage model, DEP2 genotypes were grouped into dichotomous variablessuch that carriers of a DEP2 risk allele (heterozygous or homozygous)were compared to non-carriers. Following the conditionality of DEP2linkage on carriage of the HTR1A -1019G allele, this genotype wassimilarly included in statistical models as a dichotomous variable. Sexand all first-order interaction terms between genotypes or betweengenotype and sex were also included in statistical models.Non-significant terms (p>0.05) were sequentially dropped fromstatistical models using a backward elimination process.

In the Mormon population, DEP2.0004 (odds ratio for the T allele 1.40,95% confidence interval 1.00-1.94) and DEP2.0007 (odds ratio for the Aallele 2.03, 95% confidence interval 0.99-4.48) were associated withmajor depressive disorder (Tables 6 and 7). For each marker, thefrequency of DEP2 allele carriage was highest among -1019G-positivecases, and approximately equal among all other groups. Additionally, thesame DEP2 alleles were both linked to and associated with majordepression in the Mormon population. There was also a significantDEP2.0004 genotype-by-sex interaction. In an Ashkenazi Jewishpopulation, DEP2.0004 (odds ratio for the T allele 0.59, 95% confidenceinterval 0.35-0.99) and DEP2.0006 (odds ratio for the A allele 0.43, 95%confidence interval 0.24-0.75) were associated with major depressivedisorder (Tables 8 and 9). For each marker, the frequency of DEP2 allelecarriage was lowest among -1019G-positive cases, and approximately equalamong all other groups. There was no association of DEP2.0006 in theMormon population (Table 10), or of DEP2.0007 in the Jewish population(Table 11), with major depressive disorder.

The DEP2 polymorphisms associated with major depressive disorder differbetween Mormon and Jewish populations, and opposite alleles at DEP2.0004were associated with major depressive order between the two populations.This sort of situation is not unusual in psychiatric genetics, in factit has been observed for most of the genes that have been linked toschizophrenia (Harrison and Weinberger, Mol. Psych. 10:40-68 (2005)).The most parsimonious explanation for these results is that functionalalleles of DEP2 arose on different haplotypes in the Mormon and Jewishpopulations.

TABLE 1 Microsatellite Marker HLOD Conditional HLOD D10S1656 0.8 1.9D10S2322 0.7 1.9 D10S575 3.1 4.4 D10S214 3.4 4.2 D10S1703 2.9 3.8D10S1782 2.7 3.6 D10S1222 2.7 3.5 D10S1727 2.7 3.5 D10S1676 3.3 3.6D10S1439 2.7 3.1 D10S1134 2.5 3.0 D10S1248 2.8 2.3 D10S505 2.4 1.6D10S1770 1.9 1.6 D10S1651 1.8 1.7 D10S590 1.7 1.7 D10S212 2.3 1.9D10S1711 2.0 1.8 D10S1700 2.0 1.8

TABLE 2 Marker Name Alleles Location in SEQ ID NO: 1 DEP2.0001 C, T 2955DEP2.0002 A, C 3005 DEP2.0003 C, A 33241 DEP2.0004 C, T 38048 DEP2.0005G, A 38215 DEP2.0006 G, A 77402 DEP2.0007 G, A 77333 DEP2.0007 C, T79906

TABLE 3 Marker Name Linked Chromosomes Control Chromosomes P DEP2.000110/22  5/60 0.0004 DEP2.0002 10/22  5/60 0.0004 DEP2.0003 10/22 12/580.02 DEP2.0004 13/22 17/60 0.02 DEP2.0005 13/22 17/60 0.02 DEP2.000614/22 25/60 0.09 DEP2.0007  4/22  1/60 0.02 DEP2.0008  3/22  0/60 0.02rs14663 61 17/22 34/58 0.05 rs3740013 14/22 20/60 0.003 rs4462251 12/2218/58 0.05 rs1063536  6/22  2/60 0.004 rs3781412 15/22 25/60 0.007rs1436803  7/22  6/60 0.009

TABLE 4 Marker Name Linked Chromosomes Control Chromosomes P DEP2.000210/22 45/176 0.07 DEP2.0003 10/22 32/178 0.01 DEP2.0004 13/22 61/1780.03 DEP2.0006 14/22 73/178 0.07 DEP2.0007  4/22  4/178 0.006 DEP2.0008 3/22  6/174 0.07 rs14663 61 17/22 103/176  0.11 rs3740013 14/22 75/1760.07 rs4462251 12/22 86/178 0.65 rs1063536  6/22 21/174 0.09 rs378141215/22 106/178  0.50 rs1436803  7/22 25/176 0.06

TABLE 5 Marker Name Linked Chromosomes Control Chromosomes P DEP2.000210/22 105/356 0.15 DEP2.0003 10/22 121/684 0.003 DEP2.0004 13/22 184/6860.002 DEP2.0006 14/22 251/696 0.01 DEP2.0007  4/22  17/696 0.003DEP2.0008  3/22  20/708 0.03

TABLE 6 HTR1A DEP2.0004 T+ Males Females Genotype Status Number % Number% Number % G+ Case 197/342 58 69/113 61 128/229 56 Control  85/182 4742/101 42 43/81 53 CC Case 33/77 43 20/37  54 13/40 32 Control 28/62 4511/31  35 17/31 55

TABLE 7 HTR1A DEP2.0007 A+ Genotype Status Number % G+ Case 29/347 8Control  7/183 4 CC Case 3/77 4 Control 3/63 5

TABLE 8 HTR1A DEP2.0004 T+ Genotype Status Number % G+ Case  51/132 39Control 35/64 55 CC Case 29/50 58 Control 13/21 62

TABLE 9 HTR1A DEP2.0006 A+ Genotype Status Number % G+ Case  71/132 54Control 49/65 75 CC Case 34/50 68 Control 17/22 77

TABLE 10 HTR1A DEP2.0006 A+ Genotype Status Number % G+ Case 122/347 35Control  75/183 41 CC Case 27/77 35 Control 27/63 43

TABLE 11 HTR1A DEP2.0007 A+ Genotype Status Number % G+ Case 10/132 8Control 4/65 6 CC Case 1/50 2 Control 3/22 14

Example 2 Detection of a Brain-Specific DEP2 Transcript by NorthernBlotting

Northern blotting was performed using a probe from the 3′ UTR (nt. 1295to 1713) of LHPP (SEQ ID NO:9) on a multi-tissue blot containing poly(A)RNA from the following human tissues: brain, placenta, skeletal muscle,heart, kidney, pancreas, liver, lung, spleen, and colon. The probe usedis within sequences that are common to SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, and SEQ IDNO:12.

Methods

The pre-made poly(A) RNA Northern blot was product #3140 from Ambion(Austin Tex.). PCR was conducted to amplify a product that lies entirelyin the 3′ UTR of SEQ ID NO:9 (nucleotides 1295 to 1713).

Forward primer: (SEQ ID NO: 68) GAATCTCCCAAATCCCAGAACTCA Reverse primer:(SEQ ID NO: 69) ACACCGGGCATGACACCTTCAAGT

The DNA product was labeled using an AmbionStrip-EZ DNA kit (Ambion,Austin Tex.) and [α-32P] dATP. The blot was hybridized overnight at 42degrees Celsius in ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion,Austin Tex.). The blot was washed 2×15 minutes at low stringency(2×SSPE, 0.1% SDS) and 2×15 minutes at high stringency (0.1×SSPE, 0.1%SDS). All procedures were carried out per the manufacturer'sinstructions.

Results

FIG. 28 shows the existence of at least two DEP2 transcripts. Anapproximately 1.7 kb transcript was present in approximately equalabundance across all tissues tested. An approximately 1.1 kb transcriptwas very abundant in brain and observed at low levels in skeletal muscleand lung.

Conclusion

The 1.7 kb transcript is consistent with LHPP (SEQ ID NO:9) in theliterature (Yokoi et al, J Biochem 133:607-613 (2003)). The existence ofa novel DEP2 transcript of approximately 1.1 kb was established.Furthermore, this transcript appears to be most abundant in brain.

Example 3 Observation of Enhanced DEP2 Transcript Expression onMicroarrays

Probe sets within DEP2 are present on Affymetrix U133 Plus and U133Av2microarrays. These probe sets are annotated as recognizing LHPP, howeverthey are within sequences that are common to SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, andSEQ ID NO:12.

Methods

Datasets from microarray experiments that had been conducted forunrelated purposes were mined to learn additional information regardingthe expression level of DEP2 transcripts in normal human tissues. Analpha value of 1E-12 was used for statistical significance.

Results

Data from Affymetrix U133 Plus and U133Av2 microarrays are shown inTable 11 and Table 12, respectively. Results considered statisticallysignificant are in boldface type.

TABLE 11 Tissue type Intensity p-value spinal cord (BD) 1192.5 0 univref (BD) 658.0 4.10E−30 brain (AM) 616.6 1.00E−27 brain (AM) 604.85.88E−39 Caudate nucleus (AM) 592.9 5.61E−45 basal ganglia (AM) 508.41.74E−39 hippocampus (AM) 388.4 8.36E−25 brain (AM) 323.4 1.61E−16 brain(BD) 230.5 7.06E−20 hypothalamus (AM) 228.9 8.77E−17 cerebellum (BD)198.0 4.54E−16 Adrenal gland (BD) 172.3 6.39E−09 univ ref (S) 153.51.94E−07 salivary gland (BD) 148.9 0.00013 salivary gland (BD) 128.97.24E−08 prostate (BD) 107.1 4.30E−05 testis (AM) 105.1 0.000276 retina(BD) 103.0 0.00229 ileum (AM) 100.8 0.000296 pericardium (AM) 93.50.000186 lymph node (AM) 90.6 2.71E−05 Thyroid gland (BD) 87.8 2.28E−06kidney (AM) 87.4 0.00573 Trachea (BD) 86.2 4.03E−06 aorta (AM) 84.30.000571 colon proximal (AM) 81.8 1.23E−05 liver (BD) 77.5 2.72E−06prostate (BD) 73.7 0.02 Thyroid (AM) 71.0 0.00019 fetal liver (BD) 68.60.000435 kidney (BD) 67.5 0.000257 right atrium (AM) 66.2 0.03 colondistal (AM) 65.3 0.000397 testis (BD) 59.5 0.000539 Thymus (BD) 55.10.00131 spleen (AM) 54.5 0.00491 Vena cava (AM) 53.2 0.05 jejunum (AM)47.5 0.02 uterus (BD) 46.9 0.00417 pancreas (BD) 46.2 0.03 bone marrow(BD) 45.9 0.05 Bladder (AM) 41.2 0.00923 Left ventricle (AM) 39.2 0.15left atrium (AM) 37.8 0.13 duodenum (AM) 35.0 0.04 Thymus (BD) 35.0 0.12placenta (BD) 34.3 0.01 prostate (BD) 34.1 0.03 right ventricle (AM)31.3 0.14 heart (BD) 30.8 0.08 lung (BD) 29.4 0.04 bone marrow (BD) 25.60.04 breast (AM) 24.8 0.09 Skeletal muscle (BD) 15.5 0.16 stomach (AM)14.8 0.25 ovary (AM) 14.4 0.21 pancreas (AM) 8.7 0.37 fetal brain (BD)7.4 0.32 (AM) = purchased from Ambion (BD) = purchased from BDBiosciences (S) = purchased from Sigma

TABLE 12 Tissue type Intensity p-value Frontal cortex 897.9 0 Thalamus806.1 0 Basal ganglia 634.7 0 Temporal cortex 348.3 0 Occipital cortex294.6 0 Parietal cortex 274.8 0 Medulla 257.9 0 Cerebellum 253.1 0Universal 1 83.2 0.02 Heart 47.5 0.22 Stomach 42.2 0.05 Prostate 34.20.08 Universal 2 0.7 0.49 Pancreas −20.7 0.7

All RNAs were purchased from Ambion.

Conclusion

Based on observation of statistically significant intensity data forevery central nervous system sample examined (except a single fetalbrain sample), and lack of statistically significant intensity data forany other sample examined, it appears that DEP2 transcripts arepreferentially expressed in the central nervous system. Because themicroarray probe sets are complementary to sequences common to severalnaturally occurring DEP2 transcripts, attributing intensity data fromthese probe sets specifically to LHPP may be misleading.

Example 4 Tissue Distribution of DEP2 Transcripts

The tissue distributions of human DEP2 transcripts were determined usingquantitative reverse transcription polymerase chain reaction (QPCR).Assays were conducted for each DEP2 transcript for which there was moresupportive evidence (bioinformatic or experimental) than a singleexpressed sequence tag. Because of the linkage and association of DEP2to major depressive disorder, there was particular focus on thedistributions of these transcripts in the brain.

Methods

Human total RNAs were purchased from either Ambion, Inc. (Austin, Tex.)or BD Biosciences (Franklin Lakes, N.J.).

Reverse transcription and PCR were conducted using the InvitrogenPlatinum Thermoscript One Step System qRTPCR kit following themanufacturer's instructions. 50 ng DNAse-treated total RNA was used as atemplate for each reaction. All Ct readings were normalized to 28S rRNA.A dilution series of Universal Human Reference (BD Biosciences) was usedto generate a standard curve for these analyses. Relative expressionlevels were determined by the Relative Standard Curve Method describedin the ABI Prism User Bulletin Number 2 with 28s rRNA assayed as anendogenous control for each sample. Equivalent reverse-transcriptionefficiency was assumed for gene-to-gene comparison in the absence ofquantitative standards such as purified RNA transcripts.

A schematic of DEP2 transcripts is shown in FIG. 29.

The following primers and probe were used for amplification anddetection of DEP2-1 mRNA (SEQ ID NO:2). These primers and probe do notdiscriminate against a naturally occurring splice variant of DEP2-1 (SEQID NO:7).

Set 1 (inter-exon) (SEQ ID NO: 35) 5′-CACGTACCCATCAGCCTTCAC-3′(SEQ ID NO: 36) 5′-CCTGTGGAAGGAGCATACAGT-3′ (SEQ ID NO: 37)5′-\56-FAM\CCCAGTGACGAGCACCATCCGG\36-TAMSp\-3′ (probe)Set 2 (intra-exon) (SEQ ID NO: 38) 5′-CAACACTGGCACCTGCAGAT-3′(SEQ ID NO: 39) 5′-CCACCCCATGCCATCAA-3′ (SEQ ID NO: 40)5′-\56-FAM\AAGTGGCAGAGCAGCCCCCAGC\36-TAMSp\-3′ (probe)

The following primers and probe were used for amplification anddetection of a splice variant of DEP2-1 (SEQ ID NO:5).

(SEQ ID NO: 35) 5′-CACGTACCCATCAGCCTTCAC-3′ (SEQ ID NO: 41)5′-CCCGCCTCTCCAAGACCAT-3′   (SEQ ID NO: 37)5′-\56-FAM\CCCAGTGACGAGCACCATCCGG\36-TAMSp\-3′ (probe)

The following primers and probe were used for amplification anddetection of a splice variant of DEP2-1 (SEQ ID NO:6). These primers andprobe do not discriminate against another naturally occurring splicevariant of DEP2-1 (SEQ ID NO:8).

(SEQ ID NO: 35) 5′-CACGTACCCATCAGCCTTCAC-3′ (SEQ ID NO: 42)5′-GGTACACTCATGTCCCCACCAT-3′ (SEQ ID NO: 37)5′-\56-FAM\CCCAGTGACGAGCACCATCCGG\36-TAMSp\-3′ (probe)

The following primers and probe were used for amplification anddetection of LHPP mRNA (SEQ ID NO:9).

(SEQ ID NO: 35) 5′-CACGTACCCATCAGCCTTCAC-3′ (SEQ ID NO: 43)5′-GCGCACCGGGAAGTTCAG-3′ (SEQ ID NO: 37)5′-\56-FAM\CCCAGTGACGAGCACCATCCGG\36-TAMSp\-3′ (probe)

The following primers and probe were used for amplification anddetection of a splice variant of LHPP (SEQ ID NO:12).

(SEQ ID NO: 44) 5′-TGCAAGCGATAGGAGTGGAA-3′ (SEQ ID NO: 45)5′-GGTTGTCCACGTACCCATCAG-3′ (SEQ ID NO: 46)5′-\56-FAM\CCCACCAGGCCCAGTGACGAGC\36-TAMSp\-3′ (probe)

The following primers and probe were used for amplification anddetection of a splice variant of LHPP (SEQ ID NO:20).

(SEQ ID NO: 47) 5′-GCGCACCGGGAAGTTCAG-3′ (SEQ ID NO: 48)5′-TGAAGAACAAAACAGAATGAGAATGTG-3′ (SEQ ID NO: 49)5′-\56-FAM\CCAGCTGGAGTCATTTATTCACCTTCCTTCC\36- TAMSp\-3′(probe)

The following primers and probe were used for amplification anddetection of a splice variant of LHPP (SEQ ID NO:24).

(SEQ ID NO: 50) 5′-CCACCAGTTACTTTCAGTATGAAAGCA-3′ (SEQ ID NO: 51)5′-TATCCTTTCAGAGAAGCAGCAAAAAC-3′ (SEQ ID NO: 52)5′-\56-FAM\CAGAAATGCCTGCGGCTTTTCCTG\36-TAMSp\-3′ (probe)

The following primers and probe were used for amplification anddetection of DEP2-2 (SEQ ID NO:28).

(SEQ ID NO: 53) 5′-CGCCCCAGACCCAAGAATC-3′ (SEQ ID NO: 54)5′-CAGGAAGTGCCCATCAGCCT-3′ (SEQ ID NO: 55)5′-\56-FAM\CCCGCCTCTCCAAGACCATCCCT\36-TAMSp\-3′ (probe)

The following primers and probe were used for amplification anddetection of DEP2-3 (SEQ ID NO:30).

(SEQ ID NO: 56) 5′-AGCACCATCCGGAAGTGAAG-3′ (SEQ ID NO: 57)5′-GCTGCAAGATCTGTGCATAGGA-3′ (SEQ ID NO: 58)5′-\56-FAM\CTGATGGTGAAGAGCCTGGAAGAAACCCA\36- TAMSp\-3′ (probe)

The following primers and probe were used for amplification anddetection of AK127935 (SEQ ID NO:31).

(SEQ ID NO: 59) 5′-CAATTTAGGTCGCTGCTATGGA-3′ (SEQ ID NO: 60)5′-TGGTGACTCAAAGGCCTAATGG-3′ (SEQ ID NO: 61)5′-\56-FAM\CCTGGCCTCTTAACTCATTTACCCGGG\36- TAMSp\-3′

The following primers and probe were used for amplification anddetection of AW867792 (SEQ ID NO:33).

(SEQ ID NO: 62) 5′-TGGAGGCAGCCTCGCTTTA-3′ (SEQ ID NO: 63)5′-TTGGAGGAAGAGTTCTCATGCA-3′\ (SEQ ID NO: 64)5′-\56-FAM\CCCGCAGAACCTCCACGCTGTT\36-TAMSp\-3′

Cycle threshold (Ct) values were qualitatively interpreted as follows:

Ct > 35 probably noise Ct = 30-35 possibly low abundance transcript,reliability assessed by shape of Ct curve Ct = 25-30 moderate abundancetranscript Ct = 20-25 high abundance transcript Ct < 20 very highabundance transcript

Results

Observed Ct values are shown in Table 13. For SEQ ID NO:5, onlyinter-exon results are shown.

TABLE 13 SEQ ID NO: 2 5 9 12 20 24 28 30 31 33 Adrenal gland (MP) 27.4138.66 23.27 22.54 25.68 30.16 38.01 27.7 26.68 26.64 Amygdala (AM) 20.4832 22.91 21.83 26.78 30.47 38.93 27.12 27.93 27.15 Basal ganglia (AM)19.8 36.52 22.84 21.02 26.54 30.86 37.54 26.82 27.87 26.67 Brain (AM)19.82 31.78 22.86 21.34 26.37 30.34 36.91 26.36 27.31 26.75 Brain (MP)24.5 37.02 22.91 21.99 26.01 29.9 40 26.22 26.21 25.7 Caudate nucleus(AM) 20.36 33.16 22.58 21.05 25.52 29.79 36.34 26.69 25.92 25.63 Fetalbrain (AM) 26.24 31.91 23.97 22.83 ND ND ND ND ND ND Fetal liver (MP)31.03 40 40 23.53 33.18 31.48 39.34 29.88 30.27 29.93 Globus pallidus(AM) 17.54 32.81 22.59 21 26.23 30.74 40 25.71 26.99 26.52 Heart (AM)30.96 40 26.49 25.12 40 31.59 39.7 32.78 32.72 29.93 Heart (MP) 29.5934.83 23.87 24.38 31.43 30.17 38.33 30.14 28.71 28.33 Hippocampus (AM)21.09 32.7 22.58 21.22 26.2 30.17 40 26.47 27.03 26.21 Hypothalamus (AM)19.83 28.99 22.43 20.23 26.66 30.43 33.54 25.85 27.42 26.86 Kidney (AM)31.94 32.1 23.38 21.81 31.6 30.06 35.26 29.37 30.24 29.38 Kidney (MP)27.93 38.31 22.51 21.31 26.64 30.96 39.4 26.67 27.54 27.62 Liver (AM)30.01 40 23.8 22.48 32.56 32.36 40 29.74 30.65 29.51 Liver (MP) 30.12 4023.67 22.41 29.47 31.65 40 27.96 28.59 28.12 Lung (AM) 40 32.29 25.4425.11 33.74 32.6 39.22 32.82 31.34 29.92 Lung (MP) 28.01 36.39 24.5523.66 28.05 31.7 37.84 28.46 28.39 28.21 Medulla (AM) 18.77 40 22.5 21.226.17 30.87 40 25.52 27.09 26.5 Orbital frontal cortex (AM) 18.84 30.122.47 20.92 25.86 30.12 35.83 25.65 26.96 25.94 Peripheral leukocytes(BD) 25.15 29.52 24.18 23.35 27.4 31.12 31.81 27.7 27.09 27.52 Placenta(MP) 29.52 40 25.02 24.75 33.54 31.93 38.76 35.4 29.48 28.93 Pons (AM)19.05 38.91 22.25 20.7 26.34 30.59 40 25.05 27.54 26.94 Prefrontalcortex (AM) 20.25 37.69 22.59 21.34 26.06 30.06 40 26.66 27.15 26.04Prostate (MP) 28.26 33.94 24.84 23.54 28.4 31.48 34.97 29.31 28.3 28.09Salivary gland (MP) 29.25 31.94 25.1 24.14 31.38 31.47 34.7 29.37 29.3929.02 Skeletal muscle (MP) 30.18 40 26.01 25.27 40 33.16 40 30.85 30.3329.51 Small intestine (AM) 28.42 36.4 24.03 23.19 30.37 31.34 39.5829.81 29.4 29.17 Spinal cord (AM) 18.12 31.91 21.89 20.59 27.21 31.3237.64 24.45 27.67 26.99 Spinal cord (MP) 22.88 37.62 22.34 21.29 27.431.32 39.49 24.74 27.56 27.32 Spleen (MP) 28.97 27.06 25.32 24.34 30.3632.28 29.81 29.17 28.68 28.91 Testis (MP) 25.85 40 22.65 21.96 24.4827.75 40 26.53 24 25.04 Thalamus (AM) 19.81 40 21.81 20.5 26.29 30.09 4026.28 27.33 26.48 Thymus (MP) 26.57 28.63 24.44 23.52 27.08 30.48 31.7929.56 27.04 27.26 Thyroid gland (MP) 27.56 32.25 22.87 21.72 26.23 30.0535.03 26.86 26.96 27.28 Trachea (MP) 27.85 31.73 24.44 23.53 28.36 31.1834.36 28.83 28.2 28.12 Uterus (MP) 28.04 40 24.54 23.79 28.84 32.16 4029.17 28.3 28.31 (AM) = purchased from Ambion (BD) or (MP) = purchasedfrom BD Biosciences ND = not done Data for SEQ ID NO: 2 are fromprimer/probe set 1.

DEP2-1 (SEQ ID NO:2) was detected as a very high or high abundancetranscript in all central nervous system samples tested (Ct range17.5-24.5) except for fetal brain (Ct 26.2), and as a moderate or lowabundance transcript in other tissues (Ct range 25.2-31.9) except forlung (Ct 40). Similar results were obtained using a second set ofprimers and probe within the first exon of DEP2-1 (nucleotides 1-316 ofSEQ ID NO:2).

A splice variant of DEP2-1 (SEQ ID NO:5) was reliably detected inspleen, thymus, hypothalamus and peripheral leukocytes (Ct range27.1-29.5).

A splice variant of DEP2-1 (SEQ ID NO:6) was not reliably detected (notshown).

LHPP (SEQ ID NO:9) was detected as a high or moderate abundancetranscript in all samples tested (Ct range 21.8-26.5) except fetal liver(Ct 40). Expression in central nervous system was in general slightlyhigher than in other tissues.

A splice variant of LHPP (SEQ ID NO:12) was detected as a high abundancetranscript in all samples tested (Ct range 20.2-25.2). Expression incentral nervous system was in general slightly higher than in othertissues.

A splice variant of LHPP (SEQ ID NO:20) was detected as a moderateabundance transcript in all central nervous system samples tested (Ctrange 25.5-27.4), as well as in several other tissues.

A splice variant of LHPP (SEQ ID NO:24) was detected as a moderateabundance transcript in a few samples, and as a low abundance transcriptin all others.

DEP2-2 (SEQ ID NO:28) was detected as a low abundance transcript(smallest Ct 29.8).

DEP2-3 (SEQ ID NO:30) was detected as a moderate abundance transcript inall central nervous system samples tested (Ct range 24.4-27.1), as wellas in several other tissues.

AK127935 (SEQ ID NO:31) was detected as a moderate abundance transcriptin all central nervous system samples tested (Ct range 25.9-27.9), aswell as in several other tissues.

AW867792 (SEQ ID NO:33) was detected as a moderate abundance transcriptin all samples tested (Ct range 25.0-30.0). Expression in centralnervous system was in general slightly higher than in other tissues.

Conclusions

SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:20, SEQID NO:24, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:33 arenaturally occurring transcripts arising from DEP2 (SEQ ID NO:1). Some ofthe signal observed for SEQ ID NO:5 may be attributable to SEQ ID NO:7,see Examples 5-7 below for independent experimental evidence that SEQ IDNO:5 is a naturally occurring transcript arising from DEP2. Failure todetect SEQ ID NO:6 (or SEQ ID NO:8, which would be amplified anddetected by the same primer/probe set) cannot be taken as evidence thatit is not a naturally occurring transcript arising from DEP2, withoutuse of a positive control to ensure that the assay worked. Expression oftranscripts arising from DEP2, relative to 28S rRNA, was generallyhigher in the central nervous system than in other tissues. Thedifference between central nervous system and other tissues wasstrongest for DEP2-1 (SEQ ID NO:2).

Example 5 Establishing the Sequence of DEP2-1

DEP2-1 (SEQ ID NO:2) which is a novel sequence that has never beendescribed previously, comprises distinct protein coding capacity forDep2-1a (SEQ ID NO:3) and Dep2-1b (SEQ ID NO:4), and is highly andpreferentially expressed in the central nervous system. Thesecharacteristics make it of particular interest as a candidate to explainthe linkage and association of DEP2 to major depressive disorder. DEP2-1clones were sequenced to establish whether the sequence predicted bymining EST sequence databases was correct.

Methods

IMAGE clones h3175509, h5194531, h5197955 and h4565014 were obtainedfrom the American Type Culture Collection (“ATCC”), P.O. Box 1549,Manassas, Va. 20108. DNA sequencing was performed using standard methodswell known to those practiced in the art.

Results

Aligned sequences of the four IMAGE clones and the sequence predicted byGenecarta software from EST sequences are shown in FIG. 30. Cloneh4565014 contains sequence, downstream of a polyadenylate tract, thatdoes not match DEP2.

Conclusion

The full-length sequence of DEP2-1 has been established. A novel singlenucleotide polymorphism has been identified.

Example 6 Characterization of the 5′ Ends of DEP2-1 by RLM-RACE

RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) wasperformed on a pool of human spinal cord RNA to determine the 5′ ends ofDEP2-1 (SEQ ID NO:2).

Methods

Human spinal cord total RNA (#636554 (also called 64113-1)) was obtainedfrom BD Biosciences Clontech (Palo Alto, Calif.). The FirstChoiceRLM-RACE kit and was purchased from Ambion (Austin, Tex.). The followinggene-specific RACE primers were used.

5′RACE gene specific outer primer: (SEQ ID NO: 65)TCTCCCACTGTATGCTCCTTCCA 5′RACE gene specific inner primer:(SEQ ID NO: 66) CTCTGCCACTTCATCTGCAGGT

RLM-RACE was performed using 10 μg human spinal cord total RNA accordingto manufacturer's instructions, except as noted below. Total RNA wastreated with calf intestine alkaline phosphatase to remove free5′phosphates from molecules such as rRNA, fragmented mRNA, tRNA andcontaminating DNA (this step entailed treatment with 3 μL calf intestinealkaline phosphatase (CIP) for 1.5 h at 37° C.). Full length mRNAmolecules that contain a 5′ methylated guanosine (CAP) should not havebeen affected by this treatment. The RNA was then treated with tobaccoacid pyrophosphatase to remove the CAP structure from full length mRNA,leaving a 5′phosphate. An RNA adapter oligonucleotide was ligated tofull-length mRNA using T4 RNA ligase. The adapter could not ligate todephosphorylated RNA molecules since they lacked a 5′phosphate. Arandom-primed reverse transcription reaction and nested PCR (usinggene-specific inner primers) was then used to amplify the 5′ends of aspecific transcript. As a negative control, RNA was treated with calfintestinal alkaline phosphatase but not with tobacco acidpyrophosphatease, such that T4 RNA ligase should have had no substrateto ligate to the RNA adapter oligonucleotide. RLM-RACE products wereseparated by electrophoresis, extracted from the gel, purified, andsequenced using standard methods well known to those practiced in theart.

Results

RLM-RACE was performed on 2 different lots of human spinal cord mRNA,with identical results (FIG. 31). Two bands (approximately 168 and 243nucleotides) were observed using tobacco acid pyrophosphatase-treatedRNA. These were not observed in negative control reactions.

Sequencing revealed 5′cDNA ends at nucleotides 1 and 76 of SEQ ID NO:2(FIG. 30). Clean splices at the junction between the 5′RACE Adapter andthe mRNA were not observed. This suggests that there was a mixedpopulation of DNA within a single band on the gel, and thattranscription initiation may also occur within a few bases upstream ordownstream of the major transcription start sites.

Conclusion

Two major transcription start sites of DEP2-1 have been identified.

Example 7 Characterization of the 5′ Ends of DEP2-1 by Exon-Bridging PCR

Two exon-bridging RT-PCR experiments were conducted to learn whetherDEP2-1 was a naturally occurring splice variant of LHPP, or onlyoriginates from one or more distinct transcriptional start sites. In afirst experiment, PCR was conducted using a reverse primer within thefirst exon (nucleotides 1-316) of DEP2-1 (SEQ ID NO:2) and a forwardprimer within an upstream exon of LHPP (SEQ ID NO:9). This PCR wasdesigned to amplify any transcript originating from the LHPPtranscription start and comprising exon 1 of DEP2-1. In a secondexperiment, PCR was conducted using a forward primer within an upstreamexon of LHPP and a reverse primer in the exon common to LHPP and DEP2-1.This PCR was designed to amplify any transcript containing both exons ofDEP2-1 as well as upstream sequences from LHPP.

Methods

cDNA was prepared from 3 different lots of human spinal cord mRNA (BDBiosciences Clontech) using the SuperScript III First Strand SynthesisSystem (Invitrogen, Carlsbad, Calif.). The following primers were used.

Experiment 1 Forward: (SEQ ID NO: 44) TGCAAGCGATAGGAGTGGAA Reverse:(SEQ ID NO: 39) CCACCCCATGCCATCAA Experiment 2 Forward: (SEQ ID NO: 44)TGCAAGCGATAGGAGTGGAA Reverse: (SEQ ID NO: 67) CACGTACCCATCAGCCTTCAC

PCR, electrophoresis and sequencing were performed using standardmethods well known to those practiced in the art.

Results

In experiment 1, products were observed following PCR and agarose gelelectrophoresis (FIG. 32). These were weak and not consistentlyobserved. Sequencing revealed that these were PCR artifacts notcontaining any sequence from SEQ ID NO:2. In experiment 2, products wereobserved following PCR and agarose gel electrophoresis (FIG. 33). Thiswas expected, as the primer pair used amplifies LHPP (SEQ ID NO:9). Nosequence from exon 1 (nucleotides 1-316) of SEQ ID NO:2 was detected inthese products.

Conclusion

DEP2-1 and LHPP do not share a transcriptional start site.

Example 8 Expression of LHPP, a Naturally Occurring Splice VariantThereof, and DEP2-1, in Human Neuronal and Glial Cell Lines

To establish feasibility of cell-based assays to screen for compositionsthat modulate the activity or expression of DEP2 products, quantitativePCR (“QPCR”) experiments were conducted to detect expression of DEP2-1(SEQ ID NO:2), LHPP (SEQ ID NO:9) and a naturally occurring splicevariant thereof (SEQ ID NO:12).

Methods

Cells of six ATCC cell lines (SH-SY5H, SK-N_SH, LN18, H4, Ntera2 andU87MG) were suspended in RNALater (Ambion, Austin Tex.). Total RNA wasisolated from each cell line using TRIZOL reagent (Invitrogen, CarlsbadCalif.) and purified using RNeasy columns (Qiagen, Valencia Calif.).Reverse transcription and PCR conditions were done as described inExample 4.

Results

Observed Ct values are shown in Table 14.

TABLE 14 SEQ ID NO: 2 9 12 SH-SY5H 40 40 40 SK-N_SH 31.71 24.82 23.65LN18 31.02 25.16 24.01 H4 30.04 25.79 24.72 Ntera2 29.35 24.91 23.76U87MG 31.57 27.19 26.17

Conclusions

Cell-based assays to screen for compositions that modulate expression ofSEQ ID NO:2, SEQ ID NO:9 or SEQ ID NO:12, or that modulate expression oractivity of the corresponding proteins Dep2-1a (SEQ ID NO:3), Dep2-1b(SEQ ID NO:4) or Lhpp (SEQ ID NO:10), may be feasible using five of thesix tested cell lines.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It will be readily apparentto one skilled in the art that varying substitutions and modificationsmay be made to the invention disclosed herein without departing from thescope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

1-45. (canceled)
 46. A method of screening a composition for the abilityto modulate the activity of a protein translated from SEQ ID NO:1, themethod comprising the steps of: a) providing a composition; b)simultaneously exposing the protein to the composition and to asubstrate, wherein the protein is exposed to the substrate forsufficient time and conditions to allow the substrate to react with theprotein in order to produce a reaction product or complex; and c)measuring presence or absence of said reaction product or complex,wherein a lack of said reaction product or complex indicating acomposition having the ability to modulate the activity of said protein.47. The method of claim 45, wherein the protein modifies thephosphorylation of the substrate.
 48. The method of claim 47, whereinthe protein translated from SEQ ID NO:1 has an amino acid sequenceselected from the group consisting of SEQ ID NO:10, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25 and SEQ ID NO:27.
 49. The method of claim 48, wherein thesubstrate is selected from the group consisting of: phosphohistidine,phospholysine, phosphodiimide, pyrophosphate and a peptide or proteinphosphorylated on histidine or lysine.
 50. A method of screening acomposition for the ability to modulate activity of a protein translatedfrom SEQ ID NO:1, in a cell, the method comprising the steps of: a)exposing said cell to said composition; and b) measuring the amount ofactivity of said protein in said cell, wherein a decreased or increasedamount of activity of said protein, as compared to a cell which has notbeen exposed to said composition, indicates a composition having theability to modulate the activity of said protein.
 51. The method ofclaim 45, wherein a composition having an ability to modulate theactivity of a protein can be used to treat major depression or a relateddisorder in a subject.
 52. A method of screening a composition for theability to modulate expression of a protein translated from SEQ ID NO:1,in a cell, the method comprising the steps of: a) exposing said cell tosaid composition; and b) measuring the amount of said protein in saidcell, wherein a decreased or increased amount of said protein, ascompared to a cell which has not been exposed to said composition,indicates a composition having the ability to modulate the expression ofsaid protein.
 53. A method of screening a composition for the ability tomodulate the level of expression of a protein translated from SEQ IDNO:1, the method comprising the steps of: a) exposing an in vitrotranscription and translation system comprising a regulatory sequencefrom SEQ ID NO:1 functionally connected to the open reading frame for adetectable protein, to a composition for a time and under conditionssufficient for said test whether said composition modulates the level ofexpression of the detectable protein; and b) detecting the level ofexpression of the detectable protein, wherein a reduction or an increasein the level of expression of the detectable protein indicates that saidcomposition has the ability to modulate the level of expression of aprotein translated from SEQ ID NO:1.
 54. The method of claim 52, whereinthe composition having an ability to modulate the expression of theprotein or the level of expression of the protein can be used to treatmajor depression or a related disorder in a subject.
 55. The method ofclaim 50, wherein the protein translated from SEQ ID NO:1 has an aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:32 and SEQ ID NO:34.
 56. A method of treating majordepression or a related disorder in a subject in need of said treatmentcomprising the step of administering a composition identified asmodulating the activity of a protein translated from SEQ ID NO:1, tosaid subject, in an amount sufficient to effect said treatment.
 57. Themethod of claim 56, wherein said composition inhibits or reduces theactivity of the protein.
 58. The method of claim 56, wherein saidcomposition increases the activity of the protein.
 59. A method oftreating major depression or a related disorder in a subject in need ofsaid treatment comprising reducing the amount of a protein translatedfrom SEQ ID NO:1 in said subject, to a level sufficient to effect saidtreatment.
 60. The method of claim 59, wherein said reduction resultsfrom complete binding or partial binding of a composition to saidprotein.
 61. A method of treating major depression or a related disorderin a subject in need of said treatment comprising increasing the amountof a protein translated from SEQ ID NO:1, to a level sufficient toeffect said treatment.
 62. The methods of claim 56, wherein said methodinvolves administering to said subject a therapeutically effectiveamount of a protein translated from SEQ ID NO:1.
 63. The method of claim56, wherein the protein translated from SEQ ID NO:1 has an amino acidsequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19,SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,SEQ ID NO:32 and SEQ ID NO:34. 64-74. (canceled)